Synthetic MRNA leaders

ABSTRACT

The present invention provides a synthetic mRNA leader for enhancing the expression of a gene encoding a desired protein, vectors comprising said synthetic mRNA leader and methods of producing a desired gene product using said synthetic mRNA leader and vector, said method comprising expressing said gene using a synthetic mRNA leader which comprises from 5′ to 3′: (i) a first mRNA leader sequence element; (ii) a spacer region; and (iii) a second mRNA leader sequence element; wherein said first mRNA leader sequence element is a modified transcription-stimulating mRNA leader capable of enhancing transcription of a gene relative to an unmodified reference mRNA leader sequence and/or said second mRNA leader element is a modified translation-stimulating mRNA leader capable of enhancing the translation of a gene transcript relative to an unmodified reference mRNA leader sequence.

The present invention relates generally to the fields of syntheticbiology, heterologous gene expression and protein production. Theinvention is particularly concerned with enhancing the expression (i.e.transcription and translation) of genes, particularly heterologousgenes, i.e. to improve the production of a desired protein in arecombinant gene expression system. Accordingly, the invention providesa synthetic mRNA leader for enhancing the expression of a gene encodinga desired protein, said leader comprising two mRNA leader elements,namely at least one of a transcription-stimulating element and atranslation-stimulating element, wherein said elements are separated bya spacer region. The invention also provides vectors comprising saidsynthetic mRNA leader and methods of producing a desired gene productusing said synthetic mRNA leader and vector. Further methods of theinvention include methods of identifying said transcription-stimulatingand translation-stimulating elements and optimizingtranslation-stimulating elements. In particular, thetranscription-stimulating and translation-stimulating elements may beproduced by mutating mRNA leader sequences.

The mechanisms underlying gene expression have been extensively studiedin many organisms due to their fundamental importance for theunderstanding of cell function and for application in biotechnology. Itis particularly important to have an understanding of the mechanismsaffecting expression in recombinant protein production to establishwhich factors may affect the level of expression.

It is well known in the art that protein production occurs through twobasic steps, namely transcription (to form mRNA from the DNA template)and translation (of the mRNA to form a protein). Transcription can bedelineated into three phases—initiation, elongation and termination.Hence, initiation of transcription begins with the binding of RNApolymerase to the promoter and ends with the conversion of the DNA andenzyme into an elongation complex. In between these steps, thepolymerase and promoter undergo a series of alterations that includepromoter binding and activation and RNA chain initiation and promoterescape. Promoter binding has been extensively studied in bothprokaryotes and eukaryotes, where the interactions between RNApolymerase with general transcription factors, promoter specific factorsand DNA sequences of the recognition regions of promoters have beeninvestigated. The promoter binding-activation phase leads to theformation of the open promoter complex which interacts with NTPsubstrates to initiate transcription. Short RNA transcripts can thenusually form which can be elongated if the polymerase escapes thepromoter and moves downstream.

Promoter escape is the last stage of transcription initiation where theRNA polymerase should leave the promoter region and advance todownstream regions. If the RNA polymerase has a poor ability to escapethe promoter, then abortive transcripts may be produced. Hence, theinitial transcribing complexes carry out repeated initiation andabortive release without promoter escape. In vitro studies have shownthat changes in the promoter recognition region (from −60 to −1) mayaffect the abortive rate, probability and size of abortive transcripts.

Hence, changes in the promoter and its recognition region have beenstudied in the art. Particularly, since the promoter plays an importantpart in the control of transcription, mutations in the promoter regionhave been previously studied to determine their effect on geneexpression. For example, mutations in the Pm promoter at the −10 regionwhich lies upstream of the transcriptional start site may facilitategene-independent enhancement or reduction of expression and/or improvedregulatory control of recombinant gene expression (WO 00/68375).

Translation of mRNA into protein occurs by interaction of mRNA with aribosome. At least three different types of interactions between themRNA and ribosome are known to occur. The protein moiety of the 30Ssubunit has an affinity for RNA, enabling binding in a non-sequencespecific manner. Secondly, the 3′ end of 16S rRNA interacts with a shortstretch of complementary nucleotides, known as the Shine-Dalgarnosequence, located upstream from most natural initiation codons in the 5′untranslated region. Finally, the anti-codon of fMet-tRNA pairs with theinitiation codon.

It is well known that in bacteria the efficiency of ribosome binding isprimarily determined by the secondary structure of the mRNA in thetranslational initiation region (the mRNA leader or 5′ untranslatedregion (5′UTR)—these terms are used interchangeably throughout thedescription). Mutations which have been made to hairpin structures inthis region have been shown to effect the expression by translation.Further, alterations to the Shine-Dalgarno sequence in the 5′untranslated region have also been suggested to affect translation. Infact, extending the Shine-Dalgarno sequence in the mRNA leader has beenshown to reduce translation, although this inhibitory effect could becounter-acted by introducing into the leader AU-rich sequences whichserve as targets for ribosomal protein 51, upstream of theShine-Dalgarno sequence. Mutations upstream of the ribosome binding sitemay also affect translational efficiency. Mutations made at or upstreamof the Shine-Dalgarno sequence may vary the stability of mRNA byalteration of its secondary structure or removal of a portion of theShine-Dalgarno sequence.

Therefore, it is well known in the art that mutations which affect thesecondary structure of the mRNA leader or the Shine-Dalgarno sequencemay affect translation.

It has also been shown that it is possible to generate 5′-UTR variants(i.e. mutants) which stimulate expression of recombinant genes both atthe transcriptional and translational level relative to the unmutated(i.e. wild-type) 5′UTR (WO2008/015447). It was concluded from thesefindings that mutant 5′-UTR sequences that enhance expression ofrecombinant genes might represent a compromise between transcriptionaland translational stimulation, and that it may not be possible toidentify a 5′-UTR sequence optimized for both these processes.

Accordingly, in the context of production systems for desired proteins(i.e. the expression of recombinant or heterologous genes) the factors(at the transcriptional and translational control levels) primarilythought to be important or determinative in the level or rate ofexpression achieved are the promoter and 5′UTR, at the transcriptionallevel. At the translational level, the 5′UTR is also known to have aneffect on gene expression.

In the context of protein production systems in bacteria, transcriptionand translation are coupled, wherein there is a physical link betweentranscript formation and transcript turnover (translation and mRNAdegradation). The translation rate is also likely to affect thetranscription rate which indirectly affects mRNA stability. Sinceinitiation is the rate limiting step during translation, which the5′-UTR is involved in, its sequence contributes to the overall geneexpression outcome. In other words, this region is one crucialcontributor to the maintenance of a balance between transcription,transcript stability and translation.

In the fields of metabolic engineering and synthetic biology, it isdesirable to be able to predictably control the levels of expression ofa heterologous gene in order to maximize protein output. However, toachieve maximal expression at the protein level, an ideal 5′-UTR shouldbe enhanced or optimized with respect to all functionalities and this isnot straightforward because transcription and translationfunctionalities overlap in the sequence. Whilst there are several insilico tools available for design of synthetic 5′-UTR sequences forefficient translation initiation (Na et al., (2010) BMC Syst Biol 4: 71and Salis et al., (2009) Nat Biotechnol 27: 946-950), because of itssequence proximity both to the promoter and coding region it has provendifficult to design optimal 5′-UTR sequences solely based ontranslational properties.

Surprisingly, the present inventors have now found that it is possibleto enhance, e.g. optimize, expression of a desired gene (i.e. to producea desired protein) in a recombinant gene expression system (moreparticularly a desired heterologous gene in a recombinant host, that isa host organism engineered to express said heterologous gene) bydesigning new synthetic 5′-UTR sequences that are extended in lengthrelative to a typical wild-type 5′UTR sequence, such as the Pm 5′UTR, toprovide enough space for the physical separation of an element that hasbeen designed to enhance transcription and an element that has beendesigned to enhance translation. The combination of two 5′-UTR DNAelements with distinct characteristics at the transcriptional andtranslational levels results in a significant and completely unexpectedsynergistic effect on expression, relative to either element alone.Hence, the invention concerns the production and use of a synthetic mRNAleader, which may be viewed as a dual mRNA leader or dual 5′UTR, i.e.comprising two mRNA leader elements. Furthermore, the inventors havedemonstrated that it is possible to optimize the translation element ofthe synthetic 5′UTR in silico predictably to enhance production of aprotein, which partially eliminates the need for screening. Moreover,successful combinations of elements that have been designed to enhancetranscription and elements that have been enhanced for translation canbe predicted to occur with high frequency. The inventors have alsodemonstrated that the use of the synthetic 5′UTR to enhance expressionof a desired, e.g. heterologous, gene is capable of functioning inmultiple organisms. Hence, the invention may be seen to provide anenhanced expression system with universal utility.

The inventors utilized the Pm/xylS promoter system from a TOL plasmid toexemplify the invention, but it will be evident that the invention isnot limited to this system. In brief, the inventors mutated the Pm 5′UTRto generate genetic elements for use in a synthetic 5′UTR comprising afirst sequence proximal to the promoter that is enhanced, e.g.optimized, with respect to transcription and a second sequence distal tothe promoter (i.e. downstream or 3′ to the first sequence) that isenhanced, e.g. optimized, for translation. To identify the mutatedsequences, new functional tools were needed. Accordingly, the inventorsalso designed two types of synthetic operons. A first operon is usefulfor screening and/or identifying sequences that primarily affecttranscription; the second operon is useful for screening and/oridentifying of mutants that affect translation.

Accordingly, in one aspect the invention can be seen to provide a methodof enhancing expression of a desired gene product in a recombinant geneexpression system, said method comprising expressing said gene using asynthetic mRNA leader which comprises from 5′ to 3′:

(i) a first mRNA leader sequence element;

(ii) a spacer region; and

(iii) a second mRNA leader sequence element;

wherein said first mRNA leader sequence element is a modifiedtranscription-stimulating mRNA leader capable of enhancing transcriptionof a gene relative to an unmodified reference mRNA leader sequenceand/or said second mRNA leader element is a modifiedtranslation-stimulating mRNA leader capable of enhancing the translationof a gene transcript relative to an unmodified reference mRNA leadersequence.

In a further aspect, the invention provides a synthetic mRNA leadersequence capable of enhancing expression of a desired gene product in arecombinant gene expression system, which comprises from 5′ to 3′:

(i) a first mRNA leader sequence element;

(ii) a spacer region; and

(iii) a second mRNA leader sequence element;

wherein said first mRNA leader sequence element is a modifiedtranscription-stimulating mRNA leader capable of enhancing transcriptionof a gene relative to an unmodified reference mRNA leader sequenceand/or said second mRNA leader element is a modifiedtranslation-stimulating mRNA leader capable of enhancing the translationof a gene transcript relative to an unmodified reference mRNA leadersequence.

In another aspect, the invention provides a method of identifying atranscription-stimulating mRNA leader (e.g. a leader sequence orelement), said method comprising:

(a) providing a test nucleotide sequence corresponding to a test mRNAleader;

(b) inserting the nucleotide sequence of (a) into a polycistronicexpression cassette comprising from 5′ to 3′:

(i) a first gene, being a desired gene and/or reporter gene that can beefficiently transcribed and translated;

(ii) a spacer region; and

(iii) a second gene, being a reporter gene,

wherein said nucleotide sequence is inserted upstream of said first geneand wherein said spacer region is suitable for ensuring that thetranslation of the said second gene is independent of the translation ofsaid first gene,

(c) expressing said polycistronic cassette, preferably in a host cell;

(d) determining the level of expression of said second gene; and

(e) selecting a transcription-stimulating mRNA leader by selecting anucleotide sequence which increases expression of said second generelative to an unmodified reference mRNA leader when used as a leaderupstream of said first gene in the polycistronic expression cassette,wherein said increased expression indicates enhanced transcription ofsaid first gene and hence that said test nucleotide sequence correspondsto a mRNA leader sequence element capable of stimulating transcription.

In a still further aspect, the invention provides a method ofidentifying a translation-stimulating mRNA leader (e.g. a leadersequence or element), said method comprising:

(a) providing a test nucleotide sequence corresponding to a test mRNAleader;

(b) inserting the nucleotide sequence of (a) into a polycistronicexpression cassette comprising from 5′ to 3′:

(i) a first gene, being a reporter gene that can be efficientlytranscribed and translated;

(ii) a spacer region; and

(iii) a second gene, being a desired gene and/or a reporter gene,wherein said nucleotide sequence is inserted downstream of said spacerregion and upstream of said second gene and wherein said spacer regionis suitable for ensuring that the translation of said second gene isindependent of the translation of said first gene,

(c) expressing said polycistronic cassette, preferably in a host cell;

(d) determining the level of expression of said second gene; and

(e) selecting a translation-stimulating mRNA leader by selecting anucleotide sequence which increases expression of said second generelative to an unmodified reference mRNA leader when used as a leaderupstream of said second gene in the polycistronic expression cassette,wherein said increased expression indicates enhanced translation of saidsecond gene and hence that said test nucleotide sequence corresponds toa mRNA leader sequence element capable of stimulating translation.

In another embodiment, the invention provides a vector for the selectionor identification of:

(A) a transcription-stimulating mRNA leader (e.g. a leader sequence orelement) for use in a synthetic mRNA leader of the invention, saidvector comprising:

(i) a promoter,

(ii) a polycistronic expression cassette comprising from 5′ to 3′:

(a) a first gene, being a desired gene and/or reporter gene that can beefficiently transcribed and translated;

(b) a spacer region; and

(c) a second gene, being a reporter gene, and

(iii) an insertion site for a DNA region corresponding to saidtranscription-stimulating mRNA leader upstream of said first gene,wherein said spacer region is suitable for ensuring that the translationof the said second gene is independent of the translation of said firstgene,

or

(B) a translation-stimulating mRNA leader (e.g. a leader sequence orelement) for use in a synthetic mRNA leader of the invention, saidvector comprising:

(i) a promoter,

(ii) a polycistronic expression cassette comprising from 5′ to 3′:

(a) a first gene, being a reporter gene that can be efficientlytranscribed and translated;

(b) a spacer region; and

(c) a second gene, being a desired gene and/or a reporter gene, and

(iii) an insertion site for a DNA region corresponding to saidtranslation-stimulating mRNA leader upstream of said second gene,

wherein said spacer region is suitable for ensuring that the translationof the said second gene is independent of the translation of said firstgene.

The invention also provides the use of a vector of the invention forscreening of transcription-stimulating mRNA leaders ortranslation-stimulating mRNA leaders for use in a synthetic mRNA leaderof the invention, which results in enhanced expression of a desiredgene.

In a still further embodiment, the invention provides a method ofoptimizing a synthetic mRNA leader of the invention for expression of adesired (e.g. heterologous) gene product, said method comprising:

(a) determining the translational initiation rate (TIR) for atranslational-stimulating mRNA leader (e.g. a leader sequence orelement) in combination with a desired gene using the ribosome bindingsite (RBS) calculator;

(b) applying the forward engineering function of the RBS calculator toincrease the TIR value;

(c) selecting a translation-stimulating mRNA leader with a higher TIRthan the initial translational-stimulating mRNA leader; and

(d)(i) modifying the sequence of the translation-stimulating mRNA leaderof the synthetic mRNA leader to correspond to the sequence of theoptimized translation-stimulating mRNA leader from (c); or

(ii) inserting the optimized translation-stimulating mRNA leader from(c) into a nucleic acid molecule to produce an optimized synthetic mRNAleader, wherein the translation-stimulating leader (e.g. element) isinserted upstream of said desired gene and downstream of atranscription-stimulating mRNA leader.

Enhancing expression refers to increasing or improving, and inparticular embodiments optimizing or maximizing, expression(transcription and translation) relative to a reference or control levelof expression, e.g. a modified mRNA leader element enhancestranscription and/or translation relative to an unmodified (reference)mRNA leader. Thus an increased amount of the desired gene product may beproduced. More particularly, an increased amount of protein is producedin the expression system. (The term “protein” is used broadly herein toinclude any protein, polypeptide or peptide encoded by the desiredgene.)

Effectively, the present invention combines two mRNA leaders, at leastone of which has been modified to enhance transcription (namely toprovide a transcription-stimulating mRNA leader element) or to enhancetranslation (namely to provide a translation-stimulating mRNA leaderelement), preferably both. The two leaders are incorporated into thesynthetic leader which can accordingly be seen to comprise two mRNAleader “elements”. A mRNA leader element thus simply refers to a nucleicacid molecule or nucleotide sequence, or a part thereof, that is capableof functioning as a mRNA leader. A mRNA leader element may be atranscription-stimulating element, i.e. a transcription-stimulating mRNAleader, or a translation-stimulating element, i.e. atranscription-stimulating mRNA leader. In particular, a mRNA leaderelement is a modified transcription- or translation-stimulating mRNAleader.

The term “modified” is used herein to denote that a mRNA leader sequencehas been selected, designed or altered (e.g. mutated), in particular soas to enhance (improve or increase etc.) transcription or translation.In other words, the ability of the leader to enhance transcription ortranslation is improved or increased, or an ability or effect of theleader to enhance transcription or translation is conferred by the“modification”. Alternatively expressed, the leader is “adapted” toenhance transcription or translation respectively.

An unmodified reference mRNA leader sequence may be a native orwild-type mRNA leader, e.g. a mRNA leader sequence derived from a gene,operon and/or expression system of a cell, virus or organism, such as aleader from a desired gene, Pm mRNA leader, a lac mRNA leader, a PT7ϕ10mRNA leader or a Ptrc mRNA leader. In other embodiments, an unmodifiedreference leader may contain sequence variation(s) over the wild-type ornative sequence, but such variations have not been introduced for thepurpose of enhancing transcription or translation and in particular donot act to enhance transcription or translation. In some embodiments, anunmodified reference mRNA leader may be an artificial mRNA leader, e.g.a designed mRNA leader. For instance, an artificial mRNA leader may bedesigned de novo based on the structural features that are known to berequired for a nucleotide sequence to function as a leader, e.g. aShine-Dalgarno sequence, and/or a randomly generated sequence that is acapable of functioning as an mRNA leader. Such an artificial mRNA leadermay also be a modified leader according to the invention, for example anartificial leader may be designed or selected to have transcription- ortranslation-stimulating properties.

A transcription-stimulating mRNA leader or leader element (alternativelya transcription-inducing element, transcription-facilitating element ora transcription-assisting element) refers to a nucleotide sequence that,when used as a mRNA leader (i.e. in the context of a gene expressionsystem comprising a promoter, mRNA leader and polypeptide codingsequence) results in a level of gene transcription which is increased ascompared to, or relative to, the level of gene transcription without thetranscription-stimulating element, and more particularly as compared to,or relative to an unmodified reference sequence, i.e. an unmodified mRNAleader sequence. In particular, an increase in the level of genetranscription may be an increase in the rate of transcription comparedto, or relative to, an unmodified mRNA leader sequence. However, it willbe clear from the discussion below that a transcription-stimulatingelement is not limited to increasing transcription, i.e. it may alsoresult in an increase in translation (or the rate of translation), e.g.relative to, compared to, an unmodified mRNA leader. In other words, insome embodiments, a transcription-stimulating element is not exclusivelya transcription-stimulating element. Alternatively viewed, in someembodiments, a transcription-stimulating element primarily results inincreased transcription as defined above.

A translation-stimulating mRNA leader or leader element (alternatively atranslation-inducing element, translation-facilitating element or atranslation-assisting element) refers to a nucleotide sequence that,when used as a mRNA leader (i.e. in the context of a gene expressionsystem comprising a promoter, mRNA leader and polypeptide codingsequence) results in a level of gene transcript translation (i.e.protein production or expression) which is increased as compared to, orrelative to, the level of gene transcript translation without thetranslation-stimulating element, and more particularly as compared to,or relative to, an unmodified reference sequence, i.e. an unmodifiedmRNA leader sequence. In particular, an increase in the level of genetranscript translation may be an increase in the rate of translationcompared to, or relative to, an unmodified mRNA leader sequence.Although this is generally found not to be the case, it is not precludedthat a translation-stimulating leader/leader element may also have aneffect in enhancing transcription. Thus analogously to the above, atranslation-stimulating leader/leader element may primarily result inincreased translation as defined above.

Thus, in some embodiments a mRNA leader element, e.g. a transcription-or translation-stimulating element is a modified mRNA leader sequencethat has been adapted, e.g. mutated, designed or selected, as comparedto, or relative to, an unmodified mRNA leader. Thus, a modified mRNAleader sequence may be a mutated, designed or selected mRNA leader that,when used as a mRNA leader results in a level of gene transcription orgene transcript translation which is increased as compared to, orrelative to, the level of gene transcription or gene transcripttranslation without the modified mRNA leader sequence, and moreparticularly as compared to, or relative to an unmodified mRNA leader.In particular, a modified mRNA leader may be a mutated leader, i.e. asequence in which one or more mutations are introduced. Accordingly, anunmodified reference mRNA leader may be viewed as an unmutated mRNAleader, i.e. a native or artificial (e.g. designed) mRNA leader in theabsence of the (introduced) mutations. Such an “unmutated” mRNA leaderwhich is used as the starting point for the mutations introducedaccording to the present invention may in some embodiments be a“wild-type” leader. Suitable examples of native or wild-type mRNAleaders include a leader from a desired gene, a Pm mRNA leader, a lacmRNA leader, a PT7ϕ10 mRNA leader or a Ptrc mRNA leader.

In some embodiments, a transcription-stimulating element may also resultin an increased level or rate of translation as defined above.

In some embodiments, an mRNA leader element, e.g. a transcription-and/or translation-stimulating element, may have a higher transcriptioninitiation rate (TIR) than a reference nucleotide sequence, e.g. anunmodified or unmutated mRNA leader. The TIR may be determined using theribosome binding site (RBS) calculator described in more detail below.In particular, the mRNA leader element, e.g. transcription- and/ortranslation-stimulating element, may have a higher TIR than a referencesequence for a particular gene, e.g. a desired gene and/or heterologousgene.

Thus, in some embodiments the transcription-stimulating element may havea TIR that is at least 1.1 fold higher than an appropriate referencesequence, e.g. the corresponding unmutated mRNA leader, such as at least1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.2 or 2.5 fold.Alternatively, viewed the TIR of the transcription-stimulating elementmay be increased by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,120, 150 or 200% relative to an appropriate reference sequence.Similarly, in some embodiments the translation-stimulating element mayhave a TIR that is at least 1.1 fold higher than an appropriatereference sequence, e.g. the corresponding unmutated mRNA leader, suchas at least 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0 or 14.0fold. Alternatively, viewed the TIR of the translation-stimulatingelement may be increased by at least 100, 200, 300, 400, 500, 600, 700,800, 900, 1000, 1100, 1200, 1300 or 1400% relative to an appropriatereference sequence. In some embodiments, the translation-stimulatingelement may be optimized, such that the TIR may be increased by at least15 fold relative to the initial translation-stimulating element. Forexample, the TIR may be increased by at least 20, 50, 100, 200, 500,1000, 2000, 3000, 5000, 10000, 20000, 50000, 100000 fold.

Thus, in some embodiments, the mRNA leader elements, e.g. transcription-and/or translation-stimulating elements may be artificial leadersequences. An artificial leader may be adapted or derived from anaturally occurring leader, e.g. it may be a leader which has beenmodified or mutated over the native form, i.e. is a derivative orvariant of a naturally occurring leader (e.g. a sequence modifiedderivative or variant) but which does not contain the mutationsaccording to the present invention (i.e. does not contain thetranscription- or translation-enhancing mutations which are introduced).In particular, any modification or mutation which the artificial leadermay contain relative to the native leader as it occurs in nature doesnot affect expression, and particularly transcription. In someembodiments, an artificial leader sequence may be a designed nucleicacid sequence or a randomly generated nucleic acid sequence, i.e. asequence that is not derived from known native or wild-type mRNA leader.In preferred embodiments, artificial mRNA leader elements, e.g.transcription- and/or translation-stimulating elements, comprisesequences with a low folding energy, i.e. sequences that do not foldreadily to form secondary structures. In some embodiments, the mRNAleader elements, e.g. transcription- and/or translation-stimulatingelements, contain a Shine-Dalgarno region as defined below.

The mRNA leader elements, e.g. transcription- and/ortranslation-stimulating elements, may each consist of nucleotidesequences of 10-150 nucleotides, such as 11-140, 12-130, 13-120, 14-110or 15-100 nucleotides. Thus, the mRNA leader elements, e.g.transcription- and/or translation-stimulating elements, may comprise 15,20, 25, 30, 35, 40, 45, 50 nucleotides, such as 16-90, 17-80, 18-70,19-60, 20-50, e.g. 20-45, 20-40 or 20-35 nucleotides.

A spacer region refers to a part or region of a nucleic acid moleculethat separates two other parts or elements of said nucleic acidmolecule, e.g. a part of a nucleic acid molecule that separates twofunctional elements of said nucleic acid molecule. A spacer region maybe any size or length suitable to achieve its function, which can bedetermined by routine analysis.

Thus, in the context of a synthetic mRNA leader of the invention, thespacer region or element functions to separate the mRNA leader elements,e.g. the transcription-stimulating element from thetranslation-stimulating element, e.g. to prevent unwanted interactionsor interference between the elements and/or to allow modularity andflexibility for later modifications to the elements, e.g. replacement ofone or more elements or further mutation of said elements. Thus, in someembodiments the spacer region may comprise at least 4 nucleotides, suchas at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25, 30, 40, 50, 100 or 200 nucleotides. For instance, the spacer regionmay comprise between 4-200, 5-150, 6-125, 7-100, 8-90, 9-80 or 10-70nucleotides.

In the context of the polycistronic operon or expression cassette usedin the methods of the invention, the spacer region or element functionsto ensure that translation of each gene in the operon is independent. Inother words, the spacer region must be of sufficient size to ensure thattranslation of the second gene in the operon (e.g. the desired geneand/or reporter gene) is only possible through de novo initiation (asopposed to translational read-through from the first gene). Thus, insome embodiments the spacer region may comprise at least 10 nucleotides,such as at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40,50, 100, 200, 300, 400, 500 or 1000 nucleotides. For instance, thespacer region may comprise between 10-1000, 15-900, 20-800, 30-700,40-600, 50-500 or 60-400 nucleotides.

A synthetic mRNA leader refers to any sequence that is capable offunctioning as a mRNA leader that is not a native mRNA leader, i.e.comprising elements (sequences) of a leader that allow the transcriptionof the gene to which it is associated and translation of the resultanttranscript, wherein said elements are not found together in nature. Inparticular, a synthetic mRNA leader of the invention comprises two mRNAleader elements, e.g. a transcription-stimulating element and atranslation-stimulating element, wherein said elements are separated bya spacer region. Accordingly, a synthetic mRNA leader may comprise mRNAleader elements, e.g. transcription- and translation-stimulatingelements, that are derived from different sources, or mRNA leaderelements, e.g. transcription- and translation-stimulating leadersequences, derived from the same source, but arranged in a mannerdifferent than that found in nature.

A synthetic mRNA leader (e.g. a dual mRNA leader) that enhancesexpression of a desired, e.g. heterologous, gene means said syntheticmRNA leader enhances transcription and translation (i.e. geneexpression) to a level or rate which is increased as compared to, orrelative to, the level or rate of gene expression without the syntheticmRNA leader, and more particularly as compared to, or relative to a mRNAleader element, e.g. a transcription-stimulating element ortranslation-stimulating element, as defined above, when used alone.Thus, in other words enhanced gene expression is gene expression whichis increased when using a synthetic mRNA leader of the invention, or putmore specifically a synthetic mRNA leader of the invention in which themRNA leader elements, e.g. transcription- and/or translation-stimulatingelements, are enhanced, as compared, or relative, to a correspondingsynthetic mRNA leader in which the mRNA leader elements, e.g.transcription- and translation-stimulating elements, are wild-type mRNAleaders, preferably wherein the mRNA leader elements, e.g.transcription- and translation-stimulating elements, consist of the samemRNA leader. Thus, the expression attainable with the recombinant geneexpression system according to the present invention, e.g. with apromoter and the synthetic mRNA leader, may be compared with theexpression obtained from the same expression system, but using mRNAleader elements, e.g. transcription- and translation-stimulatingelements, that are unmodified leaders rather than modified leaders, e.g.adapted, designed or selected artificial or mutant leaders. Hence, an“unmodified” or “unmutated” expression system uses the same gene andpromoter as the system where enhanced expression is seen, but the mRNAleader elements, e.g. transcription- and translation-stimulatingelements, are not modified, e.g. adapted, designed, selected or mutated.The mRNA leader used in an “unmodified” or “unmutated” expression system(i.e. reference expression system) is therefore the unmutated orunmodified mRNA leader, i.e. the “starting” leader, where nomanipulations have been carried out to enhance expression. Theunmodified leader is the leader before modification (before adaptation,mutation etc.) i.e. in embodiments where the mRNA leader elements, e.g.transcription- and translation-stimulating elements, are derived fromnative or wild-type mRNA leaders, the unmodified leader is the leaderinto which the mutations may be introduced. It may be seen as a“wild-type”, “native”, “source” or “origin” or “starting” leader or aleader which is the substrate or target for the mutations (moreparticularly, references herein to the leader include, or refer to, theDNA corresponding to the mRNA leader).

An mRNA leader, e.g. a synthetic mRNA leader of the invention, typicallyis located 3′ to (i.e. downstream of) the promoter and 5′ to (i.e.upstream of) a gene in a gene expression system or operon. In apolycistronic operon, at least one mRNA leader (e.g. the first mRNAleader in the operon) is located as defined above, wherein other mRNAleaders may be located between genes, e.g. downstream of a first geneand upstream of a second gene in said operon, downstream of a secondgene and upstream of a third gene in said operon and so on.

According to the invention, gene expression is enhanced by using acombination of mRNA leader elements, e.g. a transcription-stimulatingelement and a translation-stimulating element, wherein said elements areseparated by a spacer region. However, it will be evident from theexamples that the mRNA leader elements are capable of enhancingtranscription and/or translation independently of each other. Thus, toachieve the synergistic increase in gene expression (i.e. transcriptionand translation) it is not necessary that both elements are capableenhancing transcription and translation, respectively, relative to anunmodified reference mRNA leader. In some embodiments, thetranscription-stimulating element is capable of enhancing transcriptionof a heterologous gene relative to an unmodified mRNA leader. Inpreferred embodiments, at least the translation-stimulating element iscapable of enhancing translation of a desired, e.g. heterologous, genetranscript relative to an unmodified mRNA leader. In particularlypreferred embodiments, the transcription-stimulating element is capableof enhancing transcription of a desired, e.g. heterologous, generelative to an unmodified mRNA leader and the translation-stimulatingelement is capable of enhancing translation of a desired, e.g.heterologous, gene transcript relative to an unmodified mRNA leader.

Thus, there may be an enhancement of both gene transcription andtranslation, even if only one of the elements in the synthetic mRNAleader is enhanced relative to an unmodified mRNA leader. Thus, in someembodiments, the mRNA elements result in a synergistic (e.g. greaterthan a cumulative, cooperative or combined) increase in the level and/orrate of expression relative to an unmodified mRNA leader. Thesynergistic effect may be seen when only one of the mRNA elements isenhanced relative to an unmodified leader. In preferred embodiment, thesynergistic effect occurs when both of the mRNA elements are enhancedrelative to an unmodified leader, i.e. when the synthetic mRNA leadercontains both transcription- and translation-stimulating elements.

Whilst not wishing to be bound by theory it is hypothesized that thetranscription-stimulating element results in enhanced transcription andthe translation-stimulating element results in enhanced translation. Forinstance, when the transcription- and translation-stimulating elementsare mutated mRNA leaders, some mutations in the leaders may enhancetranslation in addition to the transcription-enhancing mutation(s)and/or the mutation which enhances transcription may itself enhancetranslation indirectly, e.g. by an increased number of transcripts beingproduced and/or directly e.g. by also affecting ribosome binding, orotherwise enhancing the process of translation. Notwithstanding this, animportant aspect of the present invention is the overall enhancement oftranscription and translation (e.g. a synergistic enhancement orcumulative, cooperative or combined enhancement) caused by coupling thetranscription- and translation-stimulating elements in a synthetic mRNAleader, which result in an increase in the amount of protein produced.

An enhancement of translation can either occur as a result of anenhancement of transcription or can be independent of transcription.Hence, an enhancement of translation which is independent oftranscription could result from, for example, more efficient ribosomebinding and the actual process of translation, rather than as a resultof more transcripts being present due to enhanced transcription. Such anenhancement of translation which is independent of transcription couldbe due to an alteration of the secondary structure of the mRNA leadersequence. An enhancement of translation which is a result of enhancedtranscription is therefore due to, for example the increased number oftranscripts being available for translation. Gene expression in thepresent invention may be enhanced by an enhancement of transcription andan enhancement of translation which is a direct result of theenhancement of transcription. However, enhancement of gene expression byan enhancement of transcription and an enhancement of translation, whichis both independent of transcription and as a direct result oftranscription, is also encompassed. It is possible, for example, thatthe transcription-stimulating element allows enhanced transcription (andenhanced translation may occur as a result of this) and that thetranslation-stimulating element improves the secondary structure of thesynthetic mRNA leader to provide enhanced (transcription independent)translation. Alternatively, it is possible thattranscription-stimulating element allows enhanced transcription (andenhanced translation which is a direct result of the enhancedtranscription) and also enhanced translation which is independent of thetranscriptional effect, e.g. by improved ribosome binding. It ispreferred in this instance that enhanced translation, which isindependent of transcription, caused by the transcription-stimulatingelement is not due to an improved secondary structure.

In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or90% of the enhanced gene expression may be caused as a result ofenhancement of transcription, e.g. as a result of the production of anincreased number of transcripts and/or an increase in the rate oftranscription. In some embodiments, at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80% or 90% of the enhanced gene expression may be as a resultof enhanced translation, e.g. as a result of improved translationinitiation and/or an increase in the rate of translation initiation.Whilst it may be possible that significant or substantially all of theenhancement of gene expression may be due to enhancement oftranscription or translation, it is generally observed that enhancementof transcription is combined with an enhancement of translation. Thesynergistic enhanced gene expression effect of the elements of thesynthetic mRNA leader of the invention (i.e. enhanced proteinproduction) is thought to be attributable to a combination of bothtranscriptional and translational effects (i.e. elements that enhanceboth transcription and translation, e.g. the rate of transcription andtranslation).

Transcription of a heterologous gene can be enhanced by up to, forexample, 46 fold or more when using a synthetic mRNA leader of theinvention compared to an unmutated or unmodified leader as definedabove. Translation a heterologous gene transcript can be enhanced by upto, for example, 170 fold or more when using a synthetic mRNA leader ofthe invention compared to an unmutated or unmodified leader as definedabove. However, it will be appreciated that this may vary significantly,depending upon the precise system used, and what the starting point is,for example relative to a system using a leader where only low levels ofexpression are obtained, a much higher enhancement in the amount ofprotein product obtained may be achievable.

Thus, an increase of transcription or the rate of transcription (forexample determined by the amount of transcript produced) of 50- or60-fold or more may be attainable. In other systems or under otherconditions the increase may be less. By way of example only,transcription of the gene (or the rate of transcription) may be enhancedby at least 60, 50, 40, 30, 27, 25, 24, 23, 22, 21, 20, 17, 15, 13, 10,8, 6, 4 or 2 fold in a system using a synthetic mRNA leader of theinvention compared to expression using the corresponding unmutated orunmodified mRNA leader as defined above. Alternatively viewed, theminimum level of enhancement which can be seen is 1.1 fold, whereintranscription or the rate of transcription can be enhanced by at least1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 or 1.9 fold. Transcription or the rateof transcription can be increased by at least 10, 20, 30, 40, 50, 60,70, 80, 90, or 100%. Other levels include at least 200, 300, 400 or500%. The level or rate of enhanced transcription of the heterologousgene can be measured by any convenient method known in the art. Forexample, transcription can be determined by measuring transcriptaccumulation, e.g. using Northern blotting, array technology orreal-time PCR.

In some embodiments, increased transcription may be measured or detectedby measuring protein accumulation or protein activity as discussedbelow. In other words, an increase in expression, as measured by proteinaccumulation or activity, may indicate (i.e. be indicative of) increasedor enhanced transcription, e.g. the level or rate of transcription.

Similarly, an increase of translation or the rate of translation (forexample determined by the amount of protein produced) of 180- or200-fold or more may be attainable. In other systems or under otherconditions the increase may be less. By way of example only, translationof the gene (or the rate of translation) may be enhanced by at least200, 180, 160, 140, 120, 100, 80, 60, 50, 40, 30, 27, 25, 24, 23, 22,21, 20, 17, 15, 13, 10, 8, 6, 4 or 2 fold in a system using a syntheticmRNA leader of the invention compared to expression using thecorresponding unmutated or wild-type mRNA leader as defined above.Alternatively viewed, the minimum level of enhancement which can be seenis 1.1 fold, wherein translation or the rate of translation can beenhanced by at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 or 1.9 fold.Translation or the rate of translation can be increased by at least 10,20, 30, 40, 50, 60, 70, 80, 90, or 100%. Other levels include at least200, 300, 400, 500, 1000, 5000 or 10000%. The level or rate of enhancedtranslation of the heterologous gene transcript can be measured by anyconvenient method known in the art. For example, translation can bedetermined by measuring protein accumulation, protein activity (i.e. theactivity of the expressed protein), wherein the levels of proteinactivity obtained using the synthetic mRNA leader as opposed to theunmodified mRNA leader are increased or enhanced. Alternatively, theamount of protein produced can be measured to determine the level ofenhanced expression (i.e. transcription and/or translation), for exampleby Western blotting or other antibody detection systems, or indeed byany method of assessing or quantifying protein. Many such methods areknown in the art.

In order to identify transcription- and/or translation-stimulatingelements, such as mRNA leader mutants which stimulate or enhancetranscription and/or translation (i.e. expression), the desired proteinproduct can be expressed with a tag or as a fusion protein, e.g. a histag or other suitable detection means, which can allow the measurementof gene expression using one assay for all different protein products.Particularly preferred as a method of identifying transcription- and/ortranslation-stimulating elements is to express the protein from apolycistronic expression cassette system (i.e. operon) defined below,where the desired gene is translationally coupled to a reporter gene.Thus, in some embodiments, the methods of the invention utilize a genewhich comprises a desired gene translationally coupled to a reportergene, i.e. to generate a fusion protein. Alternatively viewed, thepolycistronic operon may comprise a gene encoding a desired proteintranslationally coupled to a reporter gene. Preferably the gene encodingthe desired protein is provided upstream (i.e. 5′) of the reporter gene.Particularly, a reporter gene is selected whose expression levelcorrelates with the expression level of the desired gene. The levels ofexpression of the desired gene can therefore be assessed directly or anindirect indication of its expression level may be obtained by measuringthe expression level of the reporter gene which has been used. Thus, insome embodiments the level of expression of the desired gene can beassessed directly, e.g. the reporter gene may be the desired gene or thedesired gene may have a readily detectable activity akin to a reportergene.

Reporter gene expression can be determined by the activity of theprotein encoded by the reporter gene. For example, if GFP was used,levels of fluorescence obtained would correlate to the level of geneexpression of the desired gene product. Attractive reporters to use arethose whose activity or presence it is possible to quantify or assess(e.g. semi-quantitatively) efficiently or readily, particularly thosewhich result in growth or growth inhibition or cell death, as suchreporters can be readily assessed by determining cell (e.g. colony)growth or non-growth. Antibiotic resistance markers fall into thiscategory, e.g. bla encoding β-lactamase. B/a is particularly attractiveas resistance correlates well to expression level. Reporters based onactivity of the gene product may also be used, e.g. reporter genesencoding an enzyme which may produce or be involved in the production ofa detectable product or in a detectable reaction. An example of such areporter is the luc gene encoding luciferase. Such “activity-based”reporters however require individual clones to be assayed. Particularlypreferred reporter genes which can be used in the polycistronic operondescribed above (and which can be translationally coupled to the geneexpressing the desired gene product) are beta-lactamase (bla), fireflyluciferase (luc) and mCherry (a red fluorescent protein derived fromDiscosoma sp).

Increased expression of the desired gene and/or reporter gene may beused to indicate enhanced transcription and/or translation, depending onthe method. Thus, in methods of identifying a transcription-stimulatingmRNA leader (e.g. a leader sequence or element), the level of expressionof the desired and/or reporter gene (e.g. as detected by proteinaccumulation or activity) may be used to indicate an increase intranscription. Similarly, in methods of identifying atranslation-stimulating mRNA leader (e.g. a leader sequence or element),the level of expression of the desired and/or reporter gene (e.g. asdetected by protein accumulation or activity) may be used to indicate anincrease in translation.

A polycistronic operon or expression cassette refers to a region of anucleic acid that can be transcribed to produce a single mRNA thatcarries several open reading frames (ORFs), each of which is translatedindependently into a polypeptide. A dicistronic or bicistronic operon orexpression cassette refers to a nucleic acid that encodes an mRNA thatcan be transcribed to produce a single mRNA that encodes only twoproteins. In a preferred embodiment, the polycistronic operon of theinvention is a bicistronic operon.

A Shine-Dalgarno sequence may be present upstream of each ORF or gene inthe polycistronic operon. In particular, a Shine-Dalgarno sequence ispresent upstream of the gene that is not proximal to the (test)nucleotide sequence (that may be a mRNA leader element, e.g. atranscription- or translation-stimulating element) inserted into theoperon in the methods of the invention.

The first gene in the polycistronic operon of the invention must be agene that can be efficiently transcribed and translated, i.e. such thatit does not inhibit, constrain, restrict or impede the rate oftranscription of the operon, i.e. it does not introduce any undesiredrestriction on the rate of transcription. Examples of genes that can beefficiently transcribed and translated are known in the art. Forinstance, the celB gene, which encodes phosphoglucomutase, istranscribed and translated efficiently and may be used as the first genein the polycistronic operon of the invention. Any suitable gene may beused in the polycistronic operon and, in some embodiments, the firstgene used in the operon may have been modified to optimize itstranscription and/or translation. Methods of optimizing transcriptionand/or translation are known in the art, e.g. modification of sequencesto that may form secondary structures and/or codon optimization.

A method of the invention is for the production of a desired geneproduct by the expression of the gene encoding the desired product (e.g.by the expression of a heterologous gene encoding the desired proteinproduct). The present invention is thus concerned with methods ofrecombinant gene expression. As noted above, methods of recombinant geneexpression are well known in the art and have been used industrially orcommercially for the production of proteins. A variety of differentexpression systems are known and may be used to express the geneaccording to the present invention, i.e. as the basis for the presentinvention. At its most basic, an expression system includes a promoterfor expression of the desired gene and the gene it is desired toexpress, or a site for insertion of the desired gene, such that it maybe expressed under the control of the promoter. According to the presentinvention, the expression system also includes a synthetic mRNA leader,or more precisely a DNA region corresponding to the leader. Alsoincluded may be other transcriptional or translational control elementsnecessary or desirable to achieve or optimize expression, as discussedfurther below.

Accordingly, the expression system which is used to produce the desiredgene product whose expression is enhanced can be any system from which agene can be expressed, i.e. any system for the expression of a gene,more precisely for the expression of a recombinant gene. The expressionsystem may be an in vivo or in vitro system and may for example be avector, e.g. a plasmid (including e.g. phagemids or cosmids) or anartificial chromosome or a viral vector, or a construct (e.g. expressioncassette) for insertion into a vector. The vector may be autonomouslyreplicating or for chromosomal integration (e.g. a transposon-basedvector or with sites for specific or homologous recombination forintegration into the chromosome of the host cell into which the vectoris introduced). The expression system according to the inventionaccordingly comprises a promoter, preferably a strong promoter, a regioncorresponding to a synthetic mRNA leader as defined herein and a genewhich encodes the desired gene product or an insertion site for saidgene.

A vector may be introduced into a host cell, and the host cell may begrown or cultured to allow said gene to be expressed, e.g. underconditions which allow the gene to be expressed. Such expression methodsare well known in the art and widely described in the literature. Thehost cell may be any convenient or desired host cell, and may beprokaryotic or eukaryotic. Thus, all types of prokaryotic cells areincluded, most notably bacteria, and eukaryotic cells may include yeastor mammalian cells. Prokaryotic expression systems are however preferredand particularly bacterial expression systems. Accordingly the desiredgene is preferably expressed in a bacterial host cell.

The desired gene product may be a heterologous gene product. In otherwords, a heterologous gene is expressed. The gene/gene product may beheterologous to the host cell used for expression. It may also beheterologous to the promoter and/or mRNA leader element(s) used, i.e. tothe expression system. Thus, the desired gene need not be used with itsnative promoter or mRNA leader. Indeed, it is usual to design anexpression system with a promoter which is not native to the gene it isdesired to express, i.e. containing a particular promoter for expressionand in general the promoter will not be native to the gene it is desiredto express. In recombinant expression, a gene may be expressed with itsnative mRNA leader, although more usually an expression vector isdesigned to include a sequence encoding a leader for expression of thegene. According to the present invention, the synthetic mRNA leader,more particularly the mRNA leader elements, e.g. transcription- and/ortranslation-stimulating elements, need not be derived from the nativeleader of the desired gene, although this is encompassed herein, i.e.the unmodified mRNA leader may be the native leader of the desired geneand one or both mRNA leader elements may be modified leaders derived oradapted from leader of the desired gene. Thus, a synthetic mRNA leadercomprising elements derived from any mRNA leader may be used, or putmore particularly, a DNA region corresponding to such modified mRNAleader elements may be used. Thus, the elements in the regioncorresponding to the synthetic mRNA leader may be from, or may bederived from, any gene or any gene system (e.g. operon etc). The mRNAleader elements, e.g. transcription- and/or translation-stimulatingelements, may be, or may be derived or adapted from, the leader which isnative to the gene to be expressed, or it may be heterologous to thegene. It may, for example be, or may not be derived from, the unmodifiedmRNA leader (more precisely the unmodified mRNA leader-correspondingsequence) which occurs naturally with the promoter which is used forexpression, i.e. which is native to the promoter. It may alternativelybe non-native (heterologous) to both the promoter and the gene.Accordingly, the promoter and mRNA element(s) of the synthetic mRNAleader may be derived or adapted from those found naturally with thedesired gene. Alternatively viewed, one or more of the promoter, regioncorresponding to the mRNA leader element(s) and gene may not occurnaturally together. In some embodiments, for example, the one or moreleader elements of the synthetic mRNA leader may be derived from a mRNAleader which occurs naturally together with the promoter, but not withthe desired gene, i.e. the gene is heterologous, or alternatively, theone or both leader elements of the synthetic mRNA leader are derivedfrom the mRNA “native” to the gene, but not the promoter.

In the methods of identifying the transcription- ortranslation-stimulating mRNA leaders (e.g. leader sequences or elements)the test mRNA leader may be produced by modifying a mRNA leader (e.g.introducing one or more mutations into a sequence corresponding to anunmodified mRNA leader, such as a native leader or a mutant leader whichis already modified over its native form) or by generating an artificialsequence capable of functioning as a mRNA leader, e.g. by generating arandom sequence as defined above.

A preferred mRNA leader element for use according to the presentinvention is or is based on that associated with the Pm promoter. Inother words, the mRNA leader elements, e.g. transcription- and/ortranslation-stimulating elements, of the synthetic mRNA leader oridentified by the methods described herein may be based on or derived oradapted from the mRNA leader associated with the Pm promoter. Thus, the“Pm” leader is preferred to be used as the leader to be mutatedaccording to the present invention and as used herein the term “Pm mRNAleader” includes not only the native Pm mRNA leader as it occurs innature, but also derivatives or variants thereof, e.g. Pm mRNA leadersequences which have been modified over the native “original” sequence.The original Pm mRNA leader is described in Inouye et al. (Gene, 29,323-330, 1984). Pm mRNA leader derivatives or modified Pm mRNA leadersequences are described in Winther-Larsen et al. (Metabolic Engineering,2, 92-103, 2000) and WO2008/015447, which are incorporated herein byreference.

Other representative mRNA leaders include the lac leader or derivativesthereof. The leaders from the promoters PT7ϕ10 and Ptrc and derivativesthereof can also be used. However, as mentioned above, in someembodiments, the mRNA leader elements may be derived or adapted from thenative mRNA leader of the desired gene.

An expression system may contain any further elements necessary ordesirable for expression, e.g. enhancer sequences. Regulatory featuresmay also be present, e.g. start or stop codons, transcriptionalinitiators or terminators, ribosomal binding sites etc.

Further, selectable markers are also useful to include in the expressionsystems or vectors to facilitate the selection of transformants. A widerange of selectable markers are known in the art and are described inthe literature. For example, antibiotic resistance markers can be usedor the TOL plasmid Xyl E structural gene can be used. This encodes theproduct C230 which may readily be detected qualitatively or assayed.Spraying a plate of bacterial colonies with catechol rapidlydistinguishes C230⁺ colonies since they turn yellow due to theaccumulation of 2-hydroxy muconic semialdehyde, enablingtransformants/transconjugants etc rapidly to be identified by thepresence of xylE in the vectors.

As mentioned previously, in some embodiments the expression system mayalso comprise a reporter gene or tag, e.g. which may be translationallycoupled to the gene of interest. Representative reporter genes includeany antibiotic resistance gene e.g. bla, or any gene encoding adetectable product, e.g. mCherry, or an enzyme which catalyses adetectable reaction e.g. luc.

Translational coupling may be achieved using the phenomenon oftranslational reinitiation (Adkin and Van Duin, 1990, J. Mol. Biol.,213, 811-818; André et al., 2000, Febs Letters, 468, 73-78).

The expression system may conveniently be in the form of a vector, asmentioned above. As noted above, a range of vectors are possible and anyconvenient or desired vector may be used, e.g. a plasmid vector or aviral vector. A vast range of vectors and expression systems are knownin the art and described in the literature and any of these may be usedor modified for use according to the present invention. In arepresentative embodiment, vectors may be used which are based on thebroad-host-range RK2 replicon, into which an appropriate strong promotermay be introduced. For example WO 98/08958 describes RK2-based plasmidvectors into which the Pm/xylS promoter system from a TOL plasmid hasbeen introduced. Such vectors represent preferred vectors which may beused according to the present invention. Alternatively, any vectorcontaining the Pm promoter may be used, whether in plasmid or any otherform, e.g. a vector for chromosomal integration, for example atransposon-based vector. As noted above, the mRNA leader elements of thesynthetic mRNA leader may be derived from the leader of the Pm promoterand accordingly, in one representative embodiment, the Pm promoter isused with a synthetic mRNA leader comprising one or more elementsderived from the Pm mRNA leader.

Other vectors or expression systems which may be used include thosebased on or including the following promoters: Ptac, PtrcT7 RNApolymerase promoter (P₇ϕ10), λP_(L) and P_(BAD). The vectors may, asnoted above, be in autonomously replicating form, typically plasmids, ormay be designed for chromosomal integration. This may depend on the hostorganism used, for example in the case of host cells of Bacillus sp.chromosomal integration systems are used industrially, but are lesswidely used in other prokaryotes. Generally speaking for chromosomalintegration, transposon delivery vectors for suicide vectors may be usedto achieve homologous recombination. In bacteria, plasmids are generallymost widely used for protein production.

As noted above, any prokaryotic or eukaryotic cell may be used forexpression, but preferably, a prokaryotic cell. This includes both Gramnegative and Gram positive bacteria. Suitable bacteria includeEscherichia sp., Salmonella, Klebsiella, Proteus, Yersinia, Azotobactersp., Pseudomonas sp., Xanthomonas sp., Agrobacterium sp., Alcaligenessp., Bordatella sp., Haemophilus influenzae, Methylophilusmethylotrophus, Rhizobium sp., Thiobacillus sp. and Clavibacter sp. In aparticularly preferred embodiment, expression of the desired geneproduct occurs in E. coli or Pseudomonas sp., e.g. Pseudomonas putida.Eukaryotic host cells may include yeast cells or mammalian cell lines.

The desired gene product may be encoded by any desired or cloned gene,including partial gene sequences, or any nucleotide sequence encoding adesired expression product, including fusion protein products. Hence theterm “gene” refers to any nucleotide sequence which it is desired toexpress.

The gene product may be any protein it is desired to produce. The term“protein” is used broadly herein to include any protein, polypeptide orpeptide sequence. This may for example be a commercially or industriallyimportant protein. Desired gene products may thus includetherapeutically active proteins, enzymes or any protein having a usefulactivity, e.g. structural or binding proteins. Representative proteinsmay thus include enzymes involved in biosynthetic pathways or which makeor are involved in the production of any useful product. Since thepresent invention is concerned with improving the production ofcommercially or industrially useful proteins, reporter genes or reportergene products are not generally included as desired genes or desiredgene products. However, as noted above, in some embodiments a reportergene may be replaced by a desired gene, particularly when the expressionproduct of the desired gene is readily and conveniently detectable, suchthat a classic reporter gene is not required.

As used herein, the term “mRNA leader” or mRNA leader sequence isequivalent to the term “5′ untranslated region” or “UTR” and refers tothe transcribed mRNA sequence between the transcription start site andtranslation start site in mRNA. The mRNA leader sequence hence is thetranscribed sequence which begins at position +1 which relates to thetranscription start site and continues until the translation start site.The region corresponding to the mRNA leader (sequence) occurs at the DNAlevel rather than the RNA level and may therefore also be viewed as theDNA (e.g. DNA sequence or region) which encodes the leader. The regioncorresponding to the mRNA leader may thus also be seen as the DNA whichis the complement of the mRNA leader or which templates its synthesis.This is also known as the initial transcribed sequence (ITS) at the DNAlevel. Mutation of a region encoding a mRNA leader sequence can alterthe transcription start site by two to three base pairs—in such asituation, +1 will relate to the ‘new’ transcription start site andhence the synthetic mRNA leader sequence in this case will again bedefined as the sequence between +1 which relates to the transcriptionstart site and the translation start site in mRNA.

The initial transcribed sequence (ITS) occurs at the DNA level as notedabove and corresponds to or encodes the transcribed mRNA leadersequence. Hence, reference herein to introducing one or more mutationsinto a mRNA leader, refers to the mutation of the corresponding DNAsequence, i.e. the ITS sequence. Mutation of this region producescorresponding mutations in the mRNA leader sequence which is thetranscribed ITS.

A mRNA leader sequence or element or its corresponding ITS can typicallybe from 10 to 40 nucleotides long, although it may be longer (e.g. up to50, 60, 70, 80 or 100 or more nucleotides). For example, the mRNA leaderor ITS may be 30 nucleotides long, or 25, 26, 27, 28 or 29 nucleotideslong, but this may, of course, depend on the gene or promoter from whichthe mRNA leader is obtained or derived. As described above, any regionencoding an mRNA leader sequence can be used in combination with anygene to be expressed and any appropriate promoter. However, in someembodiments, when the mRNA leader elements, e.g. transcription- and/ortranslation-stimulating elements, are mutated mRNA leaders, e.g. themRNA leader that is mutated to generate one or both of said elements isnative to either the promoter or the desired gene. As noted above a PmmRNA leader sequence is preferred.

As used herein, the term “a strong promoter” refers to any strongpromoter, which allows the gene under its control to be expressed at ahigh level. The strong promoter may be naturally occurring, or it may bea modified promoter or synthetic, e.g. a derivative of a naturallyoccurring promoter. It may thus be native or non-native. The term“strong promoter” is a well-known term in the art and strong promotersare widely described in the literature. Hence, such a promoter canproduce large amounts of transcript and final protein product from thegene of interest. For example, strong promoters can express proteins ata level of at least 1% of the total cellular protein. Preferably, astrong promoter can express proteins at a level of 2, 5, 10, 15, 20, 25,30, 35, 40, 45 or 50% of the total cellular protein. In the context of asecreted or exported protein (e.g. an extracellular protein or onesupplied with a secretory sequence) levels of 1% or more, or moreparticularly of 2% or more, of total cellular protein may be viewed ashigh, and accordingly indicative of a strong promoter. For anintracellular protein, levels of 5% or 7% or more, more particularly 10%or more, may be viewed as indicative of a strong promoter. This maydepend upon the expression system, host cell and conditions used etc.Accordingly, a promoter may be a strong promoter if it achieves theabove expression levels at the selected conditions in the context of aparticular host cell and expression system, i.e. it may be a strongpromoter for the particular method and reagents used. Examples of strongpromoters are well known in the art and any such promoters can be usedin the expression system from which gene expression is enhanced. Suchpromoters for example include Pm promoter, Ptac, PtrcT7 RNA polymerasepromoter (P₇ϕ10), λP_(L) and P_(BAD) or a derivative of any aforesaidpromoter. Weak promoters are not included within the definition ofstrong promoters for the present invention and hence promoters such asP_(CON) (Dobrynin et al., Nucleic acid Res. Symp. Ser., 7, 365-376,1980) are excluded.

The promoter sequence can be found upstream of the transcription startsite and is generally viewed as covering positions for example from −60to −1, although this may vary. The promoter sequence hence does notinclude any of the transcribed sequence or the sequence at the DNA levelwhich will be transcribed. The promoter sequence does not thereforecover any of the sequence downstream of and including +1.

The present invention is particularly useful in providing a means forimproving protein production processes, particularly commercial orindustrial protein production processes. Thus, the present invention canbe used to improve, or bring up to a satisfactory orcommercially-acceptable level, expression processes which are operating(i.e. expressing the protein) at a level which is not high enough forindustrial purposes. However, as noted above, the invention may also beused to improve further processes or expression systems which arealready working efficiently, e.g. where the levels of protein producedare acceptable at an industrial or commercial level.

Accordingly, alternatively viewed, the invention provides a method ofenhancing expression of a desired gene product in a recombinant geneexpression system, wherein said gene product is produced by expressionof a gene and the expression of said gene is enhanced from an alreadyefficient expression system, said method comprising expressing said geneusing a synthetic mRNA leader as defined herein.

Furthermore, the invention provides a method of enhancing expression ofa desired gene product in a recombinant gene expression system, whereinsaid gene product is produced by expression of a gene and the expressionof said gene is enhanced from an already efficient expression system,said method comprising a method of optimizing a synthetic mRNA leaderfor the expression of a desired gene product according to the methoddescribed above and expressing said gene using said optimized syntheticmRNA leader.

The mRNA leader elements, e.g. transcription- and/ortranslation-stimulating elements, for use in the synthetic mRNA leaderof the invention may be obtained by introducing one or more mutationsinto the DNA region corresponding to the mRNA leader. The mRNA leaderelements, e.g. transcription- and/or translation-stimulating elements,that are capable of enhancing the transcription and/or translation of aheterologous gene can be identified using the methods described above.

In such alternatively viewed embodiments, an already efficient geneexpression system may be seen as one which can express proteins at alevel of at least 1% of the total cellular protein. Preferably, analready efficient expression system can express proteins at a level of2, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50% of the total cellularprotein. More particularly, for an exported or secreted protein thelevel may be 1% or more particularly 2% or more of total cellularprotein and for an intracellular protein it may be 5% or more, or moreparticularly 7 or 10% or more. The considerations in relation toconditions and systems used, as mentioned above in the context of strongpromoters, apply here also.

In embodiments in which the mRNA leader elements, e.g. transcription-and/or translation-stimulating elements, are mutated mRNA leaders,mutations can be made to the region which corresponds to the mRNA leader(i.e. to the ITS) at any one or more positions from the transcriptionstart site to the translation start site. A mutation can consist of anaddition or deletion or substitution of any one or more nucleotides inthe ITS which results in the addition or deletion or substitution of anyone or more nucleotides in the mRNA leader. Addition or deletionmutations may involve the addition or deletion of one or more basepairs. Hence, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more bases can beinserted or deleted. In a particularly preferred embodiment, however, amutation may be a substitution, which can occur at any position and mayinvolve repetition (e.g. duplication) or inversion of fragments orsegments of sequence. Hence, any of A, T(U), C or G can be substitutedwith a different base selected from A, T(U), C or G.

One or more mutations may be introduced to the ITS or mRNA leader. Theone or mutations may be a combination of substitution, addition and/ordeletion mutations or a number e.g. 2 or more additions or substitutionsor deletions. Hence, a leader or ITS can contain for example bothsubstitution and deletion mutations. Further, a leader or ITS maycontain more than one substitution mutation at different positions inthe leader. The length of the leader may also be increased, for exampleby introducing insertions or adding bases to one or both ends of theencoding sequence.

The number of mutations made is preferably in the range of 1 to 10, e.g.2, 3, 4, 5, 6, 7, 8 or 9. For example, a mRNA leader or ITS may comprise1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitution mutations, or may comprise1 substitution mutation and 1 or more (e.g. 2 or 3) deletion mutations.Alternatively, substitution and/or deletion mutations may be coupledwith mutations which extend the length of the leader.

The one or more mutations can be introduced into the ITS from position+1 i.e. the transcription start site or further downstream of thisposition. In a preferred embodiment, particularly for leaders used toproduce transcription-stimulating elements, mutations are not present atthe transcription start site or near to it, for example not withinpositions +1 to +7. Hence, mutation(s) may be present at position +8 ordownstream therefrom, for example from +8 to +40, more particularly atany one or more of positions +8, +9, +10, +11, +12, +13, +14, +15, +16,+17, +18, +19, +20, +21, +22, +23, +24, +25, +26, +27, +28, +29, +30,+31, +32, +33, +34, +35, +36, +37, +38 and/or +39. In the case of alonger or extended leader, mutations may be introduced at downstreampositions up to the length of the leader, i.e. at any one of positions+8 up to the translational start site (from +8 to the end of the ITS).As previously described, any mutation, i.e. an addition, deletion orsubstitution can be made at any of these positions. Mutations can beintroduced further downstream than position +20. For example at any oneor more of residues +21, +22, +23, +24, +25, +26, +27, +28, +29 or +30or further downstream, in the case of a longer leader. Thus, mutationscan be introduced up to the translational start site at the end of theITS.

Any such mutations may be generated by any method known in the art. Forexample, mutations may be made by mutagenesis which may be site directedor random. Random mutagenesis may be induced by chemically crosslinkingagents or by radiation, for example exposure to UV light or may involvechemical modification of the nucleotides encoding or constituting themRNA leader. Preferably mutations are introduced to the ITS sequencewhich corresponds to the mRNA leader at the DNA level. Further, the ITScan be mutated by using a ‘doped’ nucleotide mixture during itssynthesis which corresponds to the mRNA leader, where at each step inpolymerisation, the relevant wild type nucleotide is contaminated withthe three other bases. This method enables the mutation frequency to beset at any particular level.

In a particularly preferred embodiment, the mutations introduced intothe ITS or mRNA leader are non-predetermined mutations, or randommutations. Hence, the particular mutations which are introduced are notdesigned or specified before mutagenesis occurs. Thus, the mutationswhich occur are not predicted or determined. Any random mutagenesismethod known in the art can be applied to produce the non-predeterminedmutations e.g. radiation or using a ‘doped’ nucleotide mixture duringmRNA leader synthesis as already described above. The introduction ofnon-predetermined mutations preferably refers to the initial screeningstage of identifying mutations which enhance transcription and/ortranslation. Hence random mutagenesis is preferably used when producingtest sequences, e.g. in methods of identifying transcription- and/ortranslation-stimulating elements for use in the synthetic mRNA leader ofthe invention. However, once such a mutation has been identified then itcan be introduced into a mRNA leader sequence to produce atranscription- and/or translation-stimulating element by any mutagenesismethod to provide the present invention. Therefore, in this way, amutated mRNA leader can be selected as a transcription- and/ortranslation-stimulating element for use in the synthetic mRNA leader,which may be particularly suited to enhancing expression of a particulargene, e.g. in a preferred gene and promoter combination. However,mutated mRNA leaders which are found to enhance transcription and/ortranslation with one gene and/or promoter can also be used to enhancetranscription and/or translation from a different gene and/or promoter.In other words, once a transcription- and/or translation-stimulatingelement has been identified it may be used with any other transcription-and/or translation-stimulating element for any gene, although it may bepreferred to identity particular mutants for particular genes.

Further, in a preferred embodiment, the mutations introduced to theleader, particularly for leaders used to produce translation-stimulatingelements, are not made to the Shine-Dalgarno sequence and/or do notestablish or eliminate putative secondary structures. In someembodiments, particularly for leaders used to producetranscription-stimulating elements, the mutations do not include theinsertion or creation of functional AU-rich sites, e.g. ribosomalprotein binding sites (e.g. S1 binding sites), enhancer elements orU-rich sequences. In other embodiments, particularly for leaders used toproduce translation-stimulating elements, the mutations may include theinsertion or creation of functional AU-rich sites, e.g. ribosomalprotein binding sites (e.g. S1 binding sites), enhancer elements orU-rich sequences. For example the insertion or creation of AU-richtracts may or may not be included, e.g. AAGGAGGUGA (SEQ ID NO: 56),AAGGAGGU or AAGGAG.

The Shine-Dalgarno (SD) sequence is a short stretch of nucleotideslocated just upstream from most natural initiation codons with which the3′ end of 16S rRNA interacts. Usually, the Shine-Dalgarno sequencecomprises GGAG nucleotides or a similar sequence, e.g. AGGA. Excludedmutations to this sequence, particularly for leaders used to producetranslation-stimulating elements, can hence consist of substitutions tothe sequence and extending or reducing the length of the SD sequence.

Further, in a preferred embodiment, the mutations made to the leaderexclude the substitution of the entire leader sequence with a differentleader sequence. Hence, in certain embodiments where the transcription-and/or translation-stimulating elements are mutant leader sequences, atleast 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of the sequenceof the wild-type mRNA leader is retained compared to the mutatedsequence (note that the “wild-type” leader is the “unmutated” leader andhence need not be a naturally occurring leader—it may include othermodifications or may be a synthetic or artificial leader).

Alternatively viewed, where the mRNA leader elements, e.g.transcription- and/or translation-stimulating elements, are mutantleader sequences, each mutated leader sequence has at least 50%, 60%,70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to the wild-type mRNAleader sequence from which it is derived. Identity may be determinedusing the BestFit program of the Genetics Computer Group (GCG) Version10 Software package from the University of Wisconsin. The program usesthe local homology algorithm of Smith and Waterman with the defaultvalues. Gap creation penalty=8, Gap extension penalty=2, Averagematch=2.912, Average mismatch=2.003.

In another preferred embodiment, the one or more mutations which areintroduced to the mRNA leader do not alter its secondary structure, e.g.do not alter or change or create or eliminate hair pin loop or othersecondary structures.

Hence in a most preferred embodiment where the transcription- and/ortranslation-stimulating elements are mutant leader sequences, one ormore non-predetermined substitution mutations are introduced to the mRNAleader sequence but not to the Shine-Dalgarno sequence.

Although the mRNA leader elements, e.g. transcription- and/ortranslation-stimulating elements, may be derived from any mRNA leader,i.e. any mRNA leader can be mutated in the present invention; in apreferred embodiment the invention uses the mRNA leader sequence whichoccurs naturally with the Pm promoter (a “Pm mRNA leader”) whichincludes derivatives of the native sequence. Hence, in one embodimentaccording to the present invention, one or more mutations may be made tothe sequence aactagtacaataataatggagtcatgaacatatg (SEQ ID NO: 1) which isthe DNA sequence (or ITS) corresponding to a Pm leader. A representativetranscription-stimulating element, which is a mutant Pm mRNA leader, mayhave a sequence selected from SEQ ID NOs: 18-23, preferably selectedfrom any one of SEQ ID NOs: 21-23 as shown in Table 1. A representativetranslation-stimulating element, which is a mutant Pm mRNA leader, mayhave a sequence selected from SEQ ID NOs. 25-46, preferably selectedfrom any one of SEQ ID NOs: 32, 41, 42 and 45 as shown in Table 1. Thus,a synthetic mRNA leader of the invention may comprise atranscription-stimulating element selected from SEQ ID NOs. 21-23 and/ora translation-stimulating element selected from SEQ ID NOs: 32, 41, 42and 45. A particularly preferred synthetic mRNA leader may comprise atranscription-stimulating element comprising SEQ ID NO: 21 and atranslation-stimulating element comprising SEQ ID NO: 42. However, itwill be understood that these mutants were identified by screening usingparticular genes. As explained above, the effects of the mutations mayin some cases and/or to some degree be gene-specific or gene-dependent.Accordingly, whilst it may be the case that some mutants may be usefulwith different genes, particular mutants are not generally regarded tobe of universal application, and generally mutants will be selected forparticular genes.

Thus, the present invention therefore encompasses a synthetic mRNAleader sequence as defined above. Also included is the DNA sequence(ITS) corresponding to the said synthetic mRNA leader.

The Pm mRNA leaders can be mutated at any one of positions +1 to +35 andas described previously such mutations can be selected from any one ormore of a substitution mutation, a deletion mutation and an additionmutation. Preferably, positions +4 and +7 are not mutated.

Mutations are hence preferably found within the range of position +2 toposition +27, more preferably within the range from position +2 to +18,for example mutations maybe found at one or more of +2, +3, +5, +6, +8,+9, +10, +11, +12, +13, +14, +15, +16 and +17.

Vectors comprising the synthetic mRNA leader or ITS sequences of theinvention and cells and libraries comprising such vectors or thesynthetic mRNA leader sequences are also encompassed.

The synthetic mRNA leader, or more particularly the ITS, can be used toenhance expression of any gene product. However, in a preferredembodiment, the ITS can be mutated and specific mutants tailored for theenhanced expression of a particular gene may be selected. Hence, suchmutants may be identified by using them in an expression system with thedesired gene and the mutants giving the highest levels of enhancedexpression may be selected. Such mutant ITS sequences may be selectedand sequenced. These sequences, although specifically selected forenhanced expression of one gene can however still be used for theenhanced expression of other genes.

A further aspect of the present invention includes a method of obtaininga synthetic mRNA leader mutant capable of enhancing the expression of adesired gene, said method comprising:

(a) introducing one or more mutations into the DNA corresponding to asynthetic mRNA leader of the invention;

(b) selecting a synthetic mRNA leader mutant from (a) which enhancesexpression of the said desired gene.

More particularly, this aspect of the invention provides a method ofobtaining a synthetic mRNA leader mutant capable of enhancing theexpression of a desired gene, said method comprising:

(a) introducing one or more mutations into the DNA corresponding to asynthetic mRNA leader of the invention;

(b) expressing said desired gene using said synthetic mRNA leader mutantin a host cell; and

(c) selecting an synthetic mRNA leader mutant which enhances expressionof said desired gene.

Preferably the gene is desired to be expressed using a strong promoteror in an already efficient expression system and/or is a heterologousgene.

The step of introducing the mutations can be seen to generate a libraryof synthetic mRNA leader mutants (more precisely ITS mutants or mutantsof the region corresponding to the leader). This library may then bescreened to select a mutant which enhances expression of a desired gene.

The library may contain two or more mutants, preferably 3, 4, 5, 6, 8,10, 12, 15, 18, 20, 22, 25, 30, 40, 50, 100, 200, 500, 1000, 5000,10000, 50000, 100000, 200000, 500000 or more mutants.

The method of this aspect of the invention may thus be seen as a methodfor screening or identifying or selecting synthetic mRNA leader mutants.

The one or more mutations can be introduced into the ITS by any methodalready described above, although in a most preferred embodiment, one ormore mutations are introduced by using a “doped” nucleotide mixture ateach step in the polymerisation of the synthesis of one strand of asynthetic oligonucleotide covering the synthetic mRNA leader.

As described above, the methods of screening can be used to select amutant ITS which is tailored or selected for particularly high enhancedexpression for a particular gene, although such mutants can in any casethen be used to enhance expression of other gene products.

The selection of a synthetic mRNA leader or ITS mutant which can enhanceexpression of the desired gene product by enhancing transcription and/ortranslation may be carried out using methods well known in the art. Forexample, the activity of the gene product can be measured, e.g. by ELISAor a similar assay, and the activity obtained using the mutant syntheticmRNA leader can be compared to that obtained using the wild type leader.Hence, a comparison of the activity levels obtained when using both theoriginal synthetic mRNA leader and the mutant synthetic mRNA leadersequences will identify those mutants which have enhanced proteinactivity and hence gene expression. Once such enhanced expressionmutants have been identified transcriptional effects can beinvestigated, if desired, for example by determining transcript levels.Transcript levels may be measured or assessed as described above.Alternatively transcript levels may be directly assessed or determinedto select the mutants. A mutant synthetic mRNA leader or ITS can beassessed for its ability to enhance gene expression by eitherinvestigating the levels of a reporter gene product which is produced(which can either be produced on its own, or as a fusion protein withthe desired gene product, or more advantageously by translationalcoupling of reporter gene expression to the expression of the desiredgene), or by directly investigating the levels of desired gene productproduced.

Hence, in a preferred embodiment the selecting step may involve theassessment or determination of levels or the activity of a reportergene. In a particularly preferred embodiment the reporter gene is anantibiotic resistance marker e.g. bla or encodes a detectable product,e.g. mCherry, or a product which results in the production of adetectable product e.g. luc or celB. Therefore, mutant synthetic mRNAleaders which can enhance expression can be screened for example bydetecting colonies of cells transformed with the expression systemcomprising a promoter, mutant mRNA leader and reporter gene, which cangrow on media containing high concentrations of penicillin (when thereporter gene is bla) or other antibiotic. For example, a penicillinconcentration in the range 1-15 mg/ml, may be used to select highexpressers but this can be reduced by using a construct designed in aparticular way, for example, having a mutation in the Shine-Dalgarnosequence to reduce translation. This would provide a wider window foridentification of transcriptional stimulation, because addition of morethan 15 mg/ml penicillin is impractical. Alternatively, the amount ofgene product obtained with the mutant mRNA leader can be measured usingfor example Western blotting and compared to that obtained when usingthe original synthetic mRNA leader. Those mutants having enhancedexpression as defined herein are selected in accordance with the presentinvention. Such a method may not be practical for low frequency mutants.

In a further embodiment, the invention provides a method of obtaining asynthetic mRNA leader mutant which is capable of enhancing expression ofa desired gene, said method comprising the steps of:

a) introducing one or more mutations into the synthetic mRNA leadersequence of the invention;

b) producing a library comprising the mutant synthetic mRNA leadersequences upstream of the gene of interest or of a reporter gene; and

c) screening the library for synthetic mRNA leader mutants which enhanceexpression of said desired gene or reporter gene.

In this way, a library of mutated synthetic mRNA leader sequences can bescreened, wherein clones expressing protein at the required levels canbe selected using methods described above e.g. Western blotting, or byusing a reporter gene e.g. bla. By using the desired gene of interest inthe method of screening, mutant ITS sequences which are tailored oroptimum or selected for enhanced expression of that gene can beselected. If a reporter gene alone is used in the method of screening,then mutated ITS sequences which may have general application may beselected.

However, since the effects of the mutants can be gene-dependent, it ispreferred to select the mutants with reference to the desired gene.Since it would be laborious to design and construct separate expressionsystems for every desired gene, the inventors have devised a method foroptimizing the synthetic mRNA leader of the invention for the expressionof a desired gene product, as described above.

The method uses the Ribosome binding site (RBS) calculator described bySalis et al., 2009 (Nat Biotechnol 27: 946-950) and Borujeni et al.,2013 (Nucleic Acid Research, 41(4) pp. 2646-2659 andhttps://salis.psu.edu/software/) to determine the translationalinitiation rate (TIR) of the synthetic mRNA leader. Thus, the step ofdetermining the TIR preferably is based on the sequence of the wholetranslation-stimulating element in combination with initial sequence ofthe desired heterologous gene, e.g. up to 50 nucleotides for eachsequence. However, the translation-stimulating element may comprise morethan 50 nucleotides. Accordingly, the input sequence used may be up to50 nucleotides of the element from the 3′ end, e.g. up to 35, 40, 45 or50 nucleotides. The initial sequence of the desired heterologous genemay include up to the first 50 nucleotides of the desired gene from the5′ end, e.g. up to 35, 40, 45 or 50 nucleotides. However, a longer inputsequence for the translation-stimulating element and/or the desiredheterologous gene may be used in some embodiments, e.g. at least 50, 55,60, 65, 70, 80, 90 or 100 nucleotides.

Applying the forward engineering function of the RBS calculator meansthat the sequence of the translation-stimulating element may bemodified, i.e. by introducing one or more mutations as defined herein,to increase the TIR value or score, preferably to maximise the TIRvalue, for the desired gene, i.e. increase the value above the initialvalue calculated in the determining step.

Selecting a translation-stimulating element with a higher TIR value(i.e. an optimized translation-stimulating element) means selecting asequence that is calculated to have a higher TIR value than the initialinput sequence. In some embodiments, the method may include a step ofsynthesizing the selected sequence, e.g. synthesizing the sequence denovo or modifying the sequence of the initial element to generate theselected, optimized, sequence.

As discussed above, the spacer region of the synthetic mRNA leader ofthe invention may function to facilitate the modification of the leader.Accordingly, the optimized translation-stimulating element may beinserted into a nucleic acid molecule comprising a synthetic mRNAleader, e.g. to replace the translation-stimulating element, therebygenerating an optimized synthetic mRNA leader. In some embodiments, thenucleic acid molecule comprising the synthetic mRNA leader also containsthe desired heterologous gene (i.e. the nucleic acid molecule is anexpression cassette or operon), wherein the optimizedtranslation-stimulating element is inserted upstream of the desiredheterologous gene and downstream of the transcription-stimulatingelement. In some embodiments, the optimized translation-stimulatingelement is inserted into a nucleic acid molecule to produce an optimizedsynthetic mRNA leader, e.g. the desired gene can be inserted into thenucleic acid molecule later or the optimized synthetic mRNA leader maybe transferred into an operon containing the desired gene. Thus, theoptimized translation-stimulating element is inserted downstream of thetranscription-stimulating element.

In some embodiments, e.g. in methods for optimizing the synthetic mRNAleader for a desired gene, the desired (test) gene may be inserted intoan expression vector downstream of a promoter and the synthetic mRNAleader (or the insertion site for the synthetic mRNA leader) and areporter gene is inserted as a second gene in such a way that itstranslation is coupled to the translation of the upstream gene (thedesired or test gene) through overlapping or closely positioned stop andstart sites. Thus, the level of expression of the desired genedetermines the level of expression of the reporter gene. Reporter geneexpression is thus an indicator of the level of desired gene expression,and may be determined to determine desired gene expression. Convenientreporter genes to use are antibiotic resistance genes for example bla orthe kanamycin resistance gene. Any desired gene may thus be insertedinto such an operon, which may contain nucleotide sequence forselection, i.e. a potential transcription- and/ortranslation-stimulating element. A library of potential transcription-or translation-stimulating elements (e.g. mutant mRNA leaders) may begenerated in such an operon, which may be inserted into a vector, e.g. a“screening vector”. A screening vector for identifying a transcription-or translation-stimulating element preferably comprises a polycistronicoperon as defined above.

Accordingly, in a preferred embodiment, an artificially constructedoperon as defined herein can be used to screen mutant ITS/mRNA leadersequences (e.g. leader elements) in a library or otherwise. Such anoperon may be contained in any convenient vector, for example in aplasmid. Such an operon typically incorporates the desired gene whoseexpression is to be enhanced and at least one reporter gene,conveniently an antibiotic resistance marker gene e.g. bla (whichencodes beta-lactamase and confers resistance to penicillin aspreviously described). The desired gene is positioned upstream of areporter gene. In some embodiments, the desired gene may betranslationally coupled to a reporter gene. Whilst the transcription ofthe desired gene and reporter gene(s) is linked, the translation of thereporter gene (i.e. the reporter gene that is not translationallycoupled to the desired gene) is independent of the desired gene. Thevector further comprises the mutant mRNA leader element sequence andpromoter upstream of the gene of interest. Hence, the desired geneproduct is produced together with, but independently from, a reportergene and in such a way, the expression of the reporter gene can be usedto measure the expression of the desired gene.

By determining the level of reporter gene expression, the level ofdesired gene expression may be determined.

Potential mRNA leader elements, e.g. transcription- ortranslation-stimulating elements such as mRNA leader mutants, whichenhance expression of the desired gene may be determined by comparingthe level of expression (i.e. reporter gene expression) with thatobtained using the corresponding unmodified leader.

Thus to determine the level of expression, a said vector is introducedinto a host cell, and said host cell is cultured or grown to allow saiddesired and reporter genes to be expressed (e.g. under conditions whichallow said genes to be expressed).

The promoter is preferably a strong promoter.

The potential transcription- or translation-stimulating element library(e.g. mutant mRNA leader element library, i.e. test nucleotide sequencelibrary) can be made in prokaryotic cells, preferably in E. coli. Othercell types can be used to create the library, examples of which havebeen described supra. Hence, libraries can be created using for examplethe expression systems already described or the artificially constructedoperon. Such a library is plated onto agar plates, where the number oftransformants may be about 100000 or more, e.g. 200000, 300000 or more.Clones containing the artificially constructed operon can be selectedfor by antibiotic resistance, e.g. by resistance to ampicillin, wheresuch a resistance gene is also present in the operon or vectorcontaining the operon. Appropriate selectable markers have beendiscussed supra. High expression mutants can be screened for bydetecting enhanced expression of the reporter gene or the desired geneproduct and can be sequenced to identify the mutation(s) responsible forenhanced expression.

As noted above, the methods of the invention find particular utility inthe commercial or industrial production of proteins. In a preferredaspect, therefore the methods of producing a protein or of enhancingexpression of a protein relate to production-scale processes i.e. theyare carried out on a production-scale or industrial scale, rather than alaboratory experiment. The processes may be preferred in a bio-reactoror fermentor, particularly a production-scale bio-reactor or fermentor.

The invention will now be described in more detail in the followingnon-limiting Example with reference to the following drawings:

FIG. 1 shows the structure of the synthetic polycistronic operons inplasmids pAO-Tr and pAO-Tn, which are used to identify transcription-and translation-stimulating elements. Both synthetic operons (A and B)are transcribed from the inducible Pm promoter (see arrow) and containcelB (encoding phosphoglucomutase) and bla (encoding β-lactamase). SD:Shine-Dalgarno sequence. t: transcriptional terminator. The unique PciIand NdeI restriction endonuclease sites were used for the insertion ofthe degenerated oligonucleotides. A vector containing operon A isdesignated pAO-Tr and a vector containing operon B is designated pAO-Tn.Pm 5′-UTR variants identified in pAO-Tr were called Tr-UTRs and variantsidentified in pAO-Tn were named Tn-UTRs.

FIG. 2 shows 5′-UTR DNA variants identified by screening pAO-Tr- andpAO-Tn-based 5′-UTR libraries for high bla expression and positionaleffects of these variants on expression. r31 (SEQ ID NO: 21), r36 (SEQID NO: 22), r50 (SEQ ID NO: 23) are 5′-UTR variants that were identifiedfrom the Tr-UTR library (A) while n24 (SEQ ID NO: 32), n44 (SEQ ID NO:41), n47 (SEQ ID NO: 42), n58 (SEQ ID NO: 45) are candidates from theTn-UTR library (B). The wild-type (wt) UTR is set forth in SEQ ID NO: 1;LV-2 (SEQ ID NO: 18) is a control 5′-UTR variant that was previouslyshown to display transcription-stimulating abilities. Nucleotides thatwere not mutagenized are typed in capital letters. These include thePciI (ACATGT) and NdeI sites (CATATG). The putative SD sequence ishighlighted in boldface. The ATG start codon (part of the NdeI site) isunderlined. Synthetic oligonucleotides carrying different mutations wereinserted into both pAO-Tr and pAO-Tn using PciI and NdeI (FIG. 1) andtransferred to E. coli DH5a. The resulting strains harboring vectorswith Tr-UTR DNA sequences (C) or Tn-UTR DNA sequences (D) were firstgrown in liquid medium overnight, then diluted 1:10.000, and finallytransferred to agar media containing increasing ampicillinconcentrations and 0.1 mM m-toluate. This low concentration was usedinitially to make sure resistance levels were in a range allowing us todistinguish moderate phenotypic differences among clones. Results arepresented as averages of the highest ampicillin concentrations at whichgrowth was observed. Error bars point to the next tested ampicillinconcentration (at which no growth was observed).

FIG. 3 shows analysis of how combinations of variant Tr- and Tn-UTRelements affect ampicillin host tolerance in the dualUTR context.Besides the wt-UTR four different Tr- and Tn-UTRs were selected and all25 possible combinations were used in host ampicillin toleranceexperiments, as described in the legend to FIG. 2, except that here 2 mMm-toluate was used. Thirteen g L⁻¹ was the highest concentration tested,as indicated by a vertical line. Error bars point to the next testedampicillin concentration (at which no growth was observed). The UTRsequences referred to in FIG. 3 are defined in the legend for FIG. 2,above.

FIG. 4 shows β-lactamase production analysis in E. coli strainsharbouring plasmids with r31Tn- and Trn47-dualUTR combinations. (A)Recombinant E. coli DH5a strains were grown in LB until OD₆₀₀˜0.1 whenexpression was induced with 2 mM m-toluate. Five hours post inductionsamples were collected for transcript and β-lactamase activity. Thevalue for the wtwt combination was arbitrarily set to 1.0. Average andstandard deviation stem from three replicas. (B) Recombinant E. coliRV308 (ATCC31608) strains were cultivated in superbroth and induced with2 mM m-toluate at OD₆₀₀=0.6-0.8 Protein gel of cell lysates that wereseparated into the soluble (supernatant) and insoluble (pellet)fraction. Results from one representative experiment are shown. Visibleβ-lactamase bands are highlighted with a box. β-lactamase activity wasalso determined and the data corresponded to the data generated in theE. coli DH5a strains (data not shown). St: Precision Plus Dual ColorProtein standard (Bio-Rad); neg ctrl: plasmid-free E. coli RV308. TheUTR sequences referred to in FIG. 4 are defined in the legend for FIG.2, above.

FIG. 5 shows mCherry production analysis in E. coli RV308 (ATCC31608)strains harbouring plasmids with r31Tn- and Trn47-dualUTR combinations.Results from one representative experiment are shown. (A) Fluorescenceintensities were determined directly from the cultures, normalizedagainst OD₆₀₀ followed by relating all values to the values obtainedfrom strains harbouring vectors with the wtwt-dualUTR combination. Theimage at the top shows pellets from the four different cultures at theharvesting time point. (B) SDS-PAGE gel of E. coli RV308 strainsproducing mCherry. St: Precision Plus Dual Color Protein standard(Bio-Rad); neg ctrl: plasmid-free E. coli RV308. The UTR sequencesreferred to in FIG. 5 are defined in the legend for FIG. 2, above.

FIG. 6 shows a comparison of effects on expression of experimentallygenerated Tn-dualUTR DNA elements with those designed by the forwardengineering function of the RBS calculator. Results were obtained afterinduction with 0.1 mM m-toluate. (A) Ampicillin tolerance analysis. Barsindicate the highest ampicillin concentrations at which growth wasobserved. Error bars point to the next tested ampicillin concentration(at which no growth was observed). A strain harbouring a construct inwhich the short LV-2 UTR DNA sequence was inserted upstream of the blagene to relate the effects of the dualUTR sequences to those reportedpreviously for LV-2 (Berg et al., (2011) J. Biotechnol. 158: 224-230).(B) Strains producing mCherry were grown in 96-well plates. At harvest,fluorescence intensities were normalized against OD₆₀₀ to calculateaverages and standard deviations obtained from four parallel cultures.The sequences of the wt, r31 and LV-2 UTRs are defined in the legend forFIG. 2, above. The dTn1 UTR has the sequence set forth in SEQ ID NO: 50;the dTn2 UTR has the sequence set forth in SEQ ID NO: 51; the dTn3 UTRhas the sequence set forth in SEQ ID NO: 52; the dTn4 UTR has thesequence set forth in SEQ ID NO: 53; the dTn5 UTR has the sequence setforth in SEQ ID NO: 54; the dTn6 UTR has the sequence set forth in SEQID NO: 55.

FIG. 7 shows the effect on β-lactamase production of short 5′-UTR DNAsequences predicted by the RBS calculator. (A) Relative change ofampicillin tolerance of strains expressing bla coupled to DNA regionscorresponding to different 5′-UTRs (wild-type (SEQ ID NO: 1) set to 1).Results were obtained from replica plating using increasing ampicillinconcentrations and inducing expression with 2 mM m-toluate. The 5′-UTRvariants included the previously identified LV-2 variant (Berg et al.,(2009) Microb. Biotechnol. 2: 379-389; SEQ ID NO: 18) and three designed5′-UTR DNA sequences (dIB1 (SEQ ID NO: 47); dIB2 (SEQ ID NO: 48); anddIB3 (SEQ ID NO: 49)). 13 g was the highest ampicillin concentrationused. (B) Analysis of the translation initiation rates (TIR) ofdifferent UTR-b/a sequences according to the RBS calculator.

FIG. 8 shows the transfer of selected pDUTRc constructs to P. putidaKT2440 and resulting effects on mCherry production. Constructs withcombinations of dualUTR DNA elements wtwt, r31 wt, wtn47 and r31n47 weretransferred to P. putida KT2440. Recombinant strains were grown in LBmedium at 30° C. and mCherry production was induced with 1 mM m-toluate.(A) 4 hours post induction, fluorescence intensities of the differentcultures were determined, normalized against OD₆₀₀ and related to thevalues from strains harbouring the wtwt-dualUTR combination. Dataoriginate from two independent experiments. (B) SDS-PAGE of the solubleprotein fraction. St: Precision Plus Dual Color Protein standard(Bio-Rad); neg ctrl: plasmid-free P. putida KT2440. The UTR sequencesreferred to in FIG. 8 are defined in the legend for FIG. 2, above.

EXAMPLES

Materials and Methods

Bacterial Strains and Growth Conditions

Recombinant E. coli DH5α (Bethesda Research Laboratories), E. coli RV308(ATCC 31608) and P. putida KT2440 were cultivated in Lysogeny broth (LB)(10 g L⁻¹ tryptone, 5 g L⁻¹ yeast extract and 5 g L⁻¹ NaCl) or on LBagar (LB broth with 15 g L⁻¹ agar) supplemented with 0.05 g L⁻¹kanamycin unless stated otherwise. Selection of E. coli DH5αtransformants was performed at 37° C., while 30° C. was used for allgrowth experiments. Induction of the XylS/Pm system was accomplished byaddition of varying m-toluate (3-methylbenzoate) concentrations.

DNA Manipulations

DNA fragments were extracted from agarose gels using the QIAquick® gelextraction kit and from liquids using the QIAquick® PCR purification kit(QIAGEN). Plasmid DNA was isolated using the Wizard® Plus SV MiniprepsDNA purification kit (Promega) or the NucleoBond® Xtra Midi kit(Macherey-Nagel). Synthetic oligonucleotides were ordered fromSigma-Aldrich or Eurofins MWG Operon. Restriction cloning was performedaccording to recommendations from New England Biolabs. PCR reactionswere carried out with the Expand High Fidelity PCR System (Roche AppliedScience). E. coli strains were transformed using a modified RbCIprotocol (Promega) and P. putida KT2440 was transformed with aelectroporation protocol. Genetic constructs were confirmed bysequencing performed at Eurofins MWG Operon or GATC Biotech using primer5′-AACGGCCTGCTCCATGACAA-3′ (SEQ ID NO: 2) for pAO-Tr-, plB11-, pDUTR-and pDUTRc-based constructs and primers 5′-CTTTCACCAGCGTTTCTGGGTG-3′(SEQ ID NO: 3) or 5′-CAAGGATCTTACCGCTGTTG-3′ (SEQ ID NO: 4) forpAO-Tn-based constructs (see below).

Vector Constructions

All vectors are based on the mini-RK2 replicon (four-seven plasmidcopies per chromosome), containing the xylS/Pm expression cassette, andkanamycin resistance gene.

(i) Construction of the pAO-Tr and pAO-Tn Screening Vectors

Two vectors containing synthetic bicistronic operons were designed tofacilitate the identification of primarily transcription- ortranslation-stimulating mutations within Pm 5′-UTR DNA sequences.

pAO-Tr The bla gene was amplified from plasmid plB11 with the primers5′-GCAGGCGGAATTCTAATGAGGTCATGAACTTATGAGTATTCAACATT-3′ (SEQ ID NO: 5) and5′-CTAGAGGATCCCCGGGTACCTTTTCTACGG-3′ (SEQ ID NO: 6), introducing therestriction sites EcoRI and BamHI, and was cloned into the plB22 plasmidas EcoRI-BamHI fragment downstream of the celB gene. plB22 is aderivative of pLB11, where an EcoRI restriction site was introduceddownstream of the celB gene. This resulted in plasmid pAO-Tr.

pAO-Tn The celB gene and the DNA sequence corresponding to its 5′-UTRwere PCR amplified using primer pair 5′-ACCCCTTAGGCTTTATGCAACAgaaACAATAATAATGGAGTCATGAACtTATG-3′ (SEQ ID NO: 7) and5′-CTTTCACCAGCGTTTCTGGGTG-3′ (SEQ ID NO: 8) from the pAO-Tr plasmid. Theresulting PCR product was digested with Bsu36I and EcoRI andre-introduced into pAO-Tr using the same restriction sites leading topAO-Tn(−1). By this procedure, additional NdeI and PciI sites wereremoved (indicated by small letters). The bla gene was PCR amplifiedusing primer pair 5′-cggaattCAACATGTACAATAATaatg-3′ (SEQ ID NO: 9) and5′-AGCTAGAGGATCCCCGGGTA-3′ (SEQ ID NO: 10) and the resulting PCR productwas cloned as EcoRI-BamHI fragment into the pAO-Tn(−1) plasmid resultingin pAO-Tn.

(ii) Construction of Vectors to Characterize Different 5′-UTR Variants

Generally, 5′-UTR DNA sequences were integrated between the unique PciIand NdeI sites of plasmid plB11 as annealed pairs of forward and reversesynthetic oligonucleotides.

pDUTR was generated based on plB11 by replacing the Pm 5′-UTR DNA regionwith annealed oligonucleotides 5′-CATGTACAATAATAATGGAGTCATGAACATATCTTCATGAGCTCCATTATTATTGTATATGTACAATAATAATGGAGTCATGAACA-3′ (SEQ ID NO: 11) and5′-TATGTTCATGACTCCATTATTATTGTACATATACAATAATAATGGAGCTCATGAAGATATGTTCATGACTCCATTATTATTGTA-3′ (SEQ ID NO: 12). Restriction sites PciI(partial), SacI and NdeI (partial) are underlined.

pDUTRc contains an E. coli codon-optimized version of the mCherry gene,which was PCR-amplified using primers5′-GCTGCATATGGTTTCTAAAGGTGAAGAAG-3′ (SEQ ID NO: 13) and5′-GCTCGGATCCTTATCATTTATACAGTTCGTCCATAC-3′ (SEQ ID NO: 14) and digestedwith NdeI and BamHI to replace the bla gene in pDUTR.

pDUTR and pDUTRc Derivatives

Annealed synthetic oligonucleotides flanked by PciI and SacI (Tr-dualUTRDNA element) and SacI and NdeI (Tn-dualUTR DNA element) sticky ends,respectively, carrying mutations according to their Tr- and Tn-UTRcounterparts were inserted into pDUTR or pDUTRc using the appropriaterestriction enzymes. Combined, the UTR variants originating from thepDUTR and pDUTRc vector variants are called TrTn-dualUTRs in which ‘Tr’and ‘Tn’ can be replaced with the name of a certain Tr- or Tn-UTRvariant, respectively. A dualUTR consisting of a wild-type Tr- and awild-type Tn-dualUTR DNA element, e.g. would be denoted as wtwt-dualUTR.

Generation and Screening of 5′-UTR Libraries Based on pAO-Tr and pAO-Tn

5′-UTR libraries were constructed in pAO-Tr and pAO-Tn by inserting thesame annealed oligonucleotides (wild-type sequence and randomly dopedsynthetic oligonucleotide mixture) between their respective NdeI andPciI restriction sites for constructing the DI-UTR library. Aftertransformation of E. coli DH5a, libraries with ˜280,000 transformants(pAO-Tr-based) and ˜370,000 transformants (pAO-Tn-based) were generated.Screening for high ampicillin tolerance was performed similar to VeeAune et al. (2009, Microb. Biotechnol. 3: 38-47), only that 0.1 mMm-toluate in combination with 2, 3 or 4 g L⁻¹ ampicillin was used forscreening the pAO-Tr-based 5′-UTR library and 0.5 mM m-toluate with 4, 5or 6 g L⁻¹ ampicillin for screening the pAO-Tn-based 5′-UTR library. Inthe latter library, constructs with multiple insertions of the 5′-UTRoligonucleotides were observed, almost exclusively isolated from thestrains tolerating the highest ampicillin concentrations. These wereexcluded from sequencing reactions by performing colony PCR using primerpair 5′-CCGGTAGCGGGACATGGG-3′ (SEQ ID NO: 15) and5′-CAAGGATCTTACCGCTGTTG-3′ (SEQ ID NO: 16). The distinct classes of Pm5′-UTR variants that were identified by screening the 5′-UTR librariesin pAO-Tr and pAO-Tn were denoted as Tr-UTRs or Tn-UTRs, respectively.

bla Expression Analysis

Ampicillin tolerance and β-lactamase enzyme activity are approximatelyproportional when bla is expressed from xylS/Pm in E. coli. Expressionof bla was mainly assessed using ampicillin tolerance testing asdescribed previously Vee Aune et al. (supra) due to the possibility toevaluate many strains in parallel using a 96-well format. For a fewselected strains, however, an enzymatic assay was performed using theprotocol described by Winther-Larsen et al. (2000 Metab. Eng. 2:92-103). For some experiments, the expression strain E. coli RV308 wasused. Recombinant E. coli RV308 strains were grown in superbroth (3.2 gL L⁻¹ peptone, 2.0 g L⁻¹ yeast extract and 0.50 g L⁻¹ NaCl). Expressionwas induced in the mid-log phase and cultures were harvested 5 hoursafter induction with 2 mM m-toluate. 0.1 g pellet (wet weight) werewashed with 0.9% NaCl and resuspended in 1.5 mL lysis buffer (25 mMTris-HCl, pH 8.0, 100 mM NaCl, 2 mM EDTA) followed by incubation with0.2 g L⁻¹ lysozyme on ice for 45 min and sonication (3 min, 35% dutycycle, 3 output control). After addition of 10 mM MgCl₂ and treatmentwith 125 U Benzonase® Nuclease (Sigma-Aldrich) for 10 min, the lysatewas centrifuged to separate the soluble supernatant fraction from thepellet. The insoluble pellet fraction was resuspended in 1.5 mL lysisbuffer. Both fractions were subjected to SDS-PAGE analysis using 12%ClearPage™ gels and ClearPAGE™ SDS-R Run buffer (C.B.S. Scientific)followed by staining with Coomassie Brilliant blue R-250 (Merck).

mCherry Production Analysis

mCherry activity was determined with an Infinite M200 Promultifunctional microplate reader (Tecan) by measuring the fluorescenceof 200 μl untreated culture with excitation and emission wavelengths of584 nm (9 nm bandwidth) and 620 nm (20 nm bandwidth), respectively, andnormalization against OD₆₀₀. Measurements were performed in duplicates.Recombinant E. coli RV308 strains harbouring pDUTRc variants were grownin LB medium and induced with 2 mM m-toluate at OD₆₀₀=0.3-0.4.Recombinant P. putida KT2440 strains were grown in LB medium at 30° C.mCherry expression was induced with 2 mM m-toluate at OD₆₀₀=0.1-0.2 andcultures were harvested 5 hours after induction. SDS-PAGE analysis wasperformed as described above for strains producing β-lactamase.

Bioinformatics Tools

Translational initiation rates (TIRs) were determined using the reverseengineering function of the RBS calculator. The sequence input for thistools consisted of the 5′-UTR DNA sequence (up to 50 nt) and the first50 nt of the bla or mcherry gene. 5′-UTRs with optimal translationalfeatures were generated applying the forward engineering function of theRBS calculator with the following constraints: First, only the DNAregion covering the region randomized by the DI-library was changed.Secondly, flanking nucleotides at the 5′- and 3′-ends should be presentso that insertion by PciI and NdeI (IB-UTR) or SacI and NdeI(Tn-dualUTR) was possible.

Example 1

Construction of Two Synthetic Operon Vectors for Identification of5′-UTR Variants Specifically Stimulating Transcription or Translation

The initial aim was to assess whether a screening method could bedeveloped which would allow us to directly identify specific mutationswithin 5′-UTRs that lead to transcriptional or translational stimulationof gene expression. We therefore constructed two screening vectorscalled pAO-Tr and pAO-Tn which were designed to identify short length(as in wild-type Pm) 5′-UTR sequences that stimulate transcription ortranslation, respectively. This was achieved by integrating a slightlydifferent synthetic bicistronic operon into each vector (FIGS. 1A and1B). Common for both operons is the arrangement of celB (encodingphosphoglucomutase) as gene one and bla (encoding β-lactamase) as genetwo. The celB gene was chosen as it can be very efficiently transcribedand translated and hence would not introduce any undesired restriction.Host tolerance to ampicillin correlates with the produced amounts ofβ-lactamase; making it easy to identify clones with the desiredphenotype. Expression of celB and bla was driven by the positivelyregulated XylS/Pm regulator/promoter system, and both operons resided ona broad-host range mini-RK2 plasmid. The spacer region between the twogenes in the operons ensured that translation of bla was only possiblethrough de novo initiation (as opposed to translational read-through).This was confirmed by the elimination of the SD sequence upstream of blaabolishing expression of β-lactamase (results not shown).

A degenerated oligonucleotide 5′-UTR mixture was used to construct a5′-UTR variant library (˜280000 clones) in the pAO-Tr operon upstream ofthe first gene, celB. It was then assumed that any observed increasedexpression of bla (detected as higher host ampicillin tolerance) wouldbe a consequence of increased transcription due to a new 5′-UTR variant.In the pAO-Tn operon, the same degenerated 5′-UTR oligonucleotidemixture was used for construction of a library (370000 clones) in whichthe oligonucleotides were inserted upstream of bla. By screening thislibrary any observed increased ampicillin tolerance was assumed to bethe result of increased de novo translation of bla as a consequence of anew 5′-UTR variant.

Example 2

Selection of Two Distinct Classes of 5′-UTR Variants by Screening of thepAO-Tr and pAO-Tn Libraries

Recombinant E. coli strains harbouring the 5′-UTR libraries were platedon agar media containing m-toluate (induces transcription from Pm) andincreasing ampicillin concentrations. From both libraries multiplecolonies were isolated that showed elevated bla expression seemingly dueto increased transcription (Tr-UTR variants) or as a consequence ofimproved translation (Tn-UTR variants) of the bla gene. Identifiedclones could grow at up to 2.5 g L⁻¹ ampicillin in the presence of a low(0.1 mM) inducer concentration. Plasmids were isolated from such clonesand the regions corresponding to the 5′-UTR were sequenced. Syntheticoligonucleotides harbouring the identified Tr- or Tn-UTR mutations werethen inserted into pAO-Tr and pAO-Tn to confirm that the initiallyobserved ampicillin tolerance levels was actually caused by the UTRmutations. In total, the screening for increased bla expression resultedin identification of five Tr- and 21 Tn-UTRs (see Table 1), among whichTr-UTRs r31, r36, r50 (FIG. 2A), and Tn-UTRs n24, n44, n47, n58 (FIG.2B) were selected for further characterization of their transcription-or translation-affecting properties. For comparison a previouslycharacterized transcription enhancing Pm 5′-UTR variant, LV-2, was alsoincluded in this study.

TABLE 1 Sequences of 5′-UTR DNA sequences identified in different screening rounds ofpAO-Tr- and pAO-Tn-based 5′-UTR libraries and resulting tolerated ampicillinconcentrations of E. coli strains harbouring these 5′-UTR variants.  SEQm-toluate ID concentration Plasmid NO: Sequence 5′->3′ [-] [0.1 mM]pAO-Tr 17 AACATGT-ACAATAATAATGGAGTCATGAACATATG 0.025 0.25 LV-2 18.......-..C......CA.....T........... 0.025 1.0 SI-r11 19.......-..C.......C................. 0.015 0.60 SIII-r28  20.......-.-T............AA........... 0.015 0.80 SIII-r31  21.......T..C..G...................... 0.025 1.0 SIII-r36  22.......-....GT....C.....A........... 0.025 1.0 SIII-r50  23.......T..........C.........T....... 0.025 1.0 pAO-Tn 24AACATGTACAATAATAATGGAGTCATGAACATATG 0.010 0.10 SI-n2 25........GTT.....-.........T........ 0.25 2.0 SI-n3 26.........T.A.C........AA........... 0.25 2.5 SI-n13 27.......G..................C........ 0.25 2.0 SI-n15 28...........C....G.........T........ 0.25 2.0 SI-n16 29............C.............A........ 0.25 1.5 SI-n17 30.......G..................T........ 0.25 2.0 SI-n18 31........A..A.G............T........ 0.25 2.0 SI-n24 32..........T.....TA........C........ 0.25 2.0 SII-n15 33..........C.....G......T........... 0.25 1.5 SII-n17 34........A.C...............T........ 0.25 2.0 SII-n23 35.............G............T........ 0.25 2.0 SII-n25 36............G.............A........ 0.25 1.0 SII-n35 37........A.......TA........C........ 0.25 1.5 SII-n39 38........-.................T........ 0.25 2.0 SII-n41 39........A..C..C..C........T........ 0.25 2.5 SII-n42 40........A.....CT..........A........ 0.25 2.0 SII-n44 41.......G.........A........C........ 0.25 2.5 SII-n47 42........AT.A.C...A.....T........... 0.25 2.5 SII-n48 43.........T...T........AG..T........ 0.25 2.0 SII-n52 44........GT...GA...........T........ 0.25 2.0 SII-n58 45..........T...C..A........AT....... 0.25 2.5 SII-n59 46.........T..T.G.TA........T........ 0.25 2.5 The values depictedcorrespond to maximal ampicillin concentrations [g L⁻¹] at which growthwas observed. LV-2 is a previously identified Pm 5′-UTR variant. SI, SIIand SIII denote different rounds of screening. No Tr-UTR variants couldbe identified in the second screening round. Shine-Dalgarno sequencesare underlined twice and the ATG start codon is written in boldface.

Initially, we wanted to investigate whether the identified Tr- andTn-UTRs would solely cause transcriptional and translationalstimulation, respectively. To study this we first inserted the Tr-UTRsin the Tn position (FIG. 2C), and Tn-UTRs in the Tr position (FIG. 2D)in the bicistronic operon. Ampicillin tolerance testing on agar mediaindicated that Tr-UTRs r31, r36, r50 in pAO-Tr caused an increase in b/aexpression (relative to wild-type) also when inserted in the Tnposition, and the same was observed for the previously identifiedtranscription-stimulating LV-2 variant. These results were somewhatsurprising, particularly since the LV-2 variant was previously found tostimulate bla transcript accumulation to nearly the same extent as theprotein product β-lactamase. Furthermore, the LV-2 transcript displayeddecay kinetics similar to that of the corresponding wild-typetranscript. Together these results indicated that LV-2 is acting almostexclusively by stimulating transcription. Even though the explanationfor the phenotypes of the Tr mutants in the Tn position are not clearthese variant sequences were later found to be very useful for design ofa new synthetic mRNA leader, i.e. a new type of UTR (see below).

For the Tn variants the phenotypes were more as expected. The observedstimulation of expression was highest for the variants n24, n44, n47 andn58 in pAO-Tn. As expected, Tn-UTRs inserted in front of celB did notyield an increase in ampicillin resistance indicating that they do notlead to increased transcription.

In addition to the phenotypic characterization, both Tr- and Tn-UTR DNAsequences were analyzed using the reverse engineering function of theRBS calculator. This tool determines a calculated translation initiationrate (TIR; see Materials and Methods) which reflects a theoreticalapproach to predict protein production levels. Based on this analysisTr-UTRs exhibited a higher TIR (1.2-2.7 times) compared to that of thewild-type 5′-UTR. The TIR values for the Tn-UTRs were notably higherincreasing the TIR of the wild-type 5′-UTR by a factor of 9.3-14.0(Table 2), further supporting that the Tn-UTRs act on translation whilethe Tr-UTRs primarily, but perhaps not exclusively act at the level oftranscription.

TABLE 2 Calculated translation initiation rates of Tr- and Tn-UTRs incombination with bla. UTR TIR wt 522.82 LV-2 2,371.77 r31 716.42 T36625.94 r50 1,407.17 n24 4,872.99 n44 7,306.38 n47 5,331.95 n58 7,306.38

Example 3

Design and Functionality Testing of a New and Extended Length 5′-UTR(i.e. Synthetic mRNA Leader) Containing Both Tr and Tn Variant Sequences

We hypothesized that a longer 5′-UTR might act much more stimulatorythan each of its two units separately. In this design we also inserted aspacer region to physically separate the Tr and Tn units, allowingmodularity and flexibility for later modifications (FIG. 3). The new UTR(here termed dualUTR or a synthetic mRNA leader) was inserted into theXylS/Pm regulator/promoter system and mini-RK2 replicon (but nosynthetic operon) as described above. Twenty-five dualUTR constructswere created by combining Tr-UTRs (one wild-type and four variants) atthe 5′-end (Tr-position) and the same for Tn-UTRs at the 3′-end(Tn-position) (FIG. 3). Recombinant E. coli strains harbouring plasmidswith each of these 25 TrTn-dualUTRs were initially subjected toampicillin tolerance testing. Among the combinations with varying Trunits and wild-type Tn unit r31 wt caused the strongest enhancement ofampicillin tolerance (3.3-fold compared to the wtwt construct) (FIG. 3).Strains harbouring the four wtTn combinations tolerated four- (wtn58) to32-times (wtn24) more ampicillin than strains with the reference wtwtconstruct. Interestingly, mutations found in the two Tr-UTR DNAelements, r31 and r50, exerted a stimulatory effect on top of the effectcaused by the mutations of the Tn-UTR DNA elements alone. All r31Tncombinations surpassed the stimulatory effects of all the wtTncombinations, and the same was true for three out of the four r50Tncombinations. The Tn-UTR n47 exhibited a stimulatory phenotype in almostall combinations tested (except r36n47), and generally the enhancementof bla expression was also stronger than the sum of expressionenhancement achieved by the individual Tr- and Tn-UTRs.

The initial characterizations described demonstrated that it wasfeasible to generate strongly improved 5′-UTR sequences by fusingprimarily transcription and translation stimulating sequences in asingle 5′-UTR. It was also encouraging that this could be shown by onlyusing a small number of Tr- and Tn-UTR variants, meaning that successfulcombinations can be predicted to occur with high frequency.

Example 4

Characterization of the Newly Designed dualUTR Sequences at theTranscript and Protein Production Level

Samples collected from E. coli strains with four dualUTR constructs,wtwt, r31 wt, wtn47, and r31n47 were subjected to relative quantitativereal-time reverse-transcription PCR (qPCR) and β-lactamase enzymaticactivity assays. The results confirmed that r31 and n47 indeed exhibitedtranscription and translation stimulating characteristics, respectively(FIG. 4A). The r31 wt UTR variant stimulated transcription 2.7-foldwhile β-lactamase activity increased only 1.7-fold. In contrast, thewtn47 dualUTR exhibited a strong translation stimulating phenotype,resulting in a 42-fold increased β-lactamase enzyme activity compared toa 10-fold increase in accumulated bla transcript relative to the wtwtdualUTR. It may well be that the transcript stimulation in this case iscaused by ribosome protection of mRNA from degradation due to the veryefficient translation. Interestingly, the r31n47 combination caused anenhancement of both processes leading to an extremely strong stimulationof expression (46- and 170-fold at the transcript and protein levels,respectively). This effect was more than the multiplicity of therelative effects the single Tr- and Tn-UTR elements had on their own.

The bla gene is generally expressed at low levels per gene copy,explaining how a 170-fold stimulation is possible. In addition a lowcopy-number plasmid (four-seven copies per chromosome) was used in theexperiments reported here. However, the very strong stimulation observedwith r31n47 suggested to us that the produced β-lactamase might in thiscase be sufficient to be directly visualized with SDS-PAGE. Totalcellular protein produced in four selected (plus negative control)bla-expressing strains was separated into the soluble and insolublefraction and analyzed on an SDS-PAGE. β-lactamase could be visualized inthe soluble fraction of sonicated cell lysates from strains harbouringconstructs with the wtn47- and r31n47 UTRs, and also in the insolublefraction from the r31n47 construct (FIG. 4B upper panel). Specificdetection of β-lactamase was also performed by Western blotting and thesignal strengths generally correlated with the protein activities (FIG.4B lower panel).

Example 5

Assessment of the Functionality of the dualUTR Design Using a DifferentReporter Gene

As the Tn-UTR variants used in the bifunctional were identified byscreening for high bla expression, it was also of interest to analyze towhat extent the stimulation was gene-specific. To assess this potentialcontext dependency, bla was substituted by mCherry, encoding a redfluorescent protein. Production of this protein was analyzed withplasmid constructs containing the wtwt, r31 wt, wtn47 and r31n47 dualUTRvariants, using a fluorimetric assay and direct protein gel analysis.The fluorescence data confirmed that the combination of thetranscription stimulating r31 and the translation stimulating n47 led tostrong synergistic effects on protein production also for mCherry,although the effects were somewhat weaker than for β-lactamase (FIG.5A). Correspondingly, the mCherry protein could be easily visualizeddirectly by SDS-PAGE both in the soluble and insoluble fraction,particularly from r31n47 (FIG. 5B). Stimulation of mCherry expression bythe r31n47 dualUTR went far beyond that of the variants r31 and n47alone, thereby confirming the corresponding observation for bla. Theseresults clearly demonstrate that there is a very significant potentialin 5′-UTR design for the improvement of recombinant gene expression.

Example 6

Use of a Rational Design Tool to Adapt the dualUTR to Different CodingRegions

The results described above showed that combination of mutations thatprimarily stimulate transcription or translation within the dualUTR gavevery good expression outcomes for at least the two tested genes, bla andmCherry. It is desirable to be able to predict theoretically thepotential Tn-UTR interactions with coding sequences. For example, some5′ proximal coding sequences may sequester the SD sequence causinginefficient translation initiation. Therefore we applied a widely usedRBS design tool, the RBS calculator, to design Tn-UTRs that are adaptedto avoid undesired interactions with the 5′ proximal end of the codingsequence. The bla and mCherry genes were again used as reporters toenable comparisons with the sequences identified by experimentalscreening. In total, six such designed Tn-UTRs were synthesized: threefor the bla coding region and three for mCherry (named, dTn-UTRs, Table3). The predicted TIR values were 60-80-fold higher compared to n47-b/a,and 50-70-fold higher compared to n47-mcherry (Table 4). All sixdTn-UTRs were inserted into the dualUTR construct with either the wt- orthe r31-variant in the Tr-position. A direct experimental comparisonwith n47, the best sequence from screening, revealed that, for bla, allthree dTn-UTRs stimulated expression to an extent that was not verydifferent from that of n47. Also, the r31-dTn1 UTR is at least as goodas r31n47 (FIG. 6A). Similar observations were also be made for mCherry(FIG. 6B). Tr-UTR r31 in combination with the designed Tn-UTRs led to 9(dTn4), 46 (dTn5) and 46 (dTn6) times relative increase vs. 58 timeswith the Tn-UTR n47. These results show that the RBS calculator can beapplied predictably to enhance production of a protein, provided the UTRdesign described here is applied.

To strengthen our observation that physical separation of the mutationswithin a 5′-UTR leading to increased transcription or translation,respectively, are necessary to improve reliability of rational designtools, we utilized the RBS calculator to design three Pm 5′-UTR variants(32 nt in length) with maximized TIR that were specific for the bla gene(dIB1-3; Table 3). When we tested the effect of these designed 5′-UTRvariants on bla expression, it became evident that optimizing a short5′-UTR for maximum TIR only is not sufficient to maximize proteinproduction (FIG. 7). Recombinant E. coli DH5α strains harbouringconstructs with the LV-2 Pm 5′-UTR variant for instance tolerated morethan 13 g L⁻¹ ampicillin while strains with the best designed 5′-UTRvariant (dIB2) only tolerated a maximum of 8 g L L⁻¹ ampicillin.

The dualUTR design is superior to the previously short optimized 5′-UTRsmainly due to two factors: (i) Higher expression levels can be achievedwith the extended length UTRs than with the improved short 5′-UTRsalone; (ii) Due to separation of the transcription and translationinfluencing regions, a Tn-UTR region can be improved solely based on itstranslation influencing characteristics, which means that sequences canbe optimized in silico.

TABLE 3  Sequences of various 5′-UTR DNA sequences.  NameSequence 5′->3′ dIB1 AACATGTTCGTCTTCACGCTAAGGAGGTACATATG (SEQ ID NO: 47) dIB2 AACATGTTACTTATACGAGGAGGTTACAGCATATG (SEQ ID NO: 48) dIB3 AACATGTACCGTTCTTTCTAAGCGAGGTTCATATG (SEQ ID NO: 49) dTn1 GAGCTCCATTATTATTGTATATGTGCATCAATTACTAAGGAGGTATACTATG (SEQ ID NO: 50) dTn2GAGCTCCATTATTATTGTATATGTGCATCACCCTT TAAGGAGGTTTACTATG (SEQ ID NO: 51)dTn3 GAGCTCCATTATTATTGTATATGTACCGTACCCGTTAAGGAGGTTTTCTATG (SEQ ID NO: 52) dTn4GAGCTCCATTATTATTGTATATGTAACAAGGCAGA ATAAGGAGGTTCATATG (SEQ ID NO: 53)dTn5 GAGCTCCATTATTATTGTATATGTGGATATACCCAGTAAGGAGGTACATATG (SEQ ID NO: 54) dTn6GAGCTCCATTATTATTGTATATGTATATAAGGATT AGAGGAGGTAATATATG (SEQ ID NO: 55)Shine-Dalgarno sequences are double-underlined and the ATG start codonis written in boldface. dIB1-3 are sequences with the same length as thePm 5′-UTR that were designed by the RBS calculator to yield the highesttranslation initiation rate possible for expression of bla. dTn1-6represent sequences of six Tn-UTR dualUTR elements with maximal TIRs forbla (dTn 1-3) or mcherry (dTn 4-6).

TABLE 4 Calculated translation initiation rates of Tn-dualUTR DNAelements in combination with bla and mcherry. dTn1-6 represent sequencesof six Tn-UTR dualUTR elements with maximal TIRs for bla (dTn 1-3) ormcherry (dTn 4-6). TIR Tn-dualUTR bla mcherry wt 598.4 2,308.6 n245,332.0 5,678.7 n44 7,994.5 25,075.0 n47 5,834.1 12,766.2 n58 3,461.44,743.2 dTn1/dTn4 349,161.5 856,820.0 dTn2/dTn5 418,029.8 819,114.1dTn3/dTn6 478,456.9 655,630.7

Example 7

Assessment of the Functionality of the Bifunctional UTR Concept in anAlternative Host

One of the great advantages of mini-RK2 replicons and the XylS/Pm systemis that they both function in Gram-negative hosts other than E. coli.One such host, Pseudomonas putida shares the same anti-SD sequencewithin the 16S rRNA with E. coli. We therefore hypothesized that thedualUTR constructs might also display similar effects on recombinantprotein production as observed in E. coli. Constructs with the TrTndualUTR combinations wtwt, r31 wt, wtn47 and r31n47 and the mCherryreporter gene were transferred to P. putida KT2440 (strain cured for theRK2 plasmid) and mCherry production was analyzed (FIG. 8A). A strongsynergistic effect of combining the r31 Tr-UTR DNA element with the n47Tn-UTR DNA element was also observed in P. putida KT2440, even thoughr31 alone had a somewhat negative effect on expression. In addition,mCherry production appeared to be more effective in this host comparedto E. coli judged by the stronger bands on the SDS-PAGE gel (FIG. 8B).The reduction of mCherry production seen for the r31 wt-compared to thewtwt UTR may potentially be attributed to a less optimal context betweenthe Pm promoter and the r31 UTR DNA sequence in this host. In any case,the data collected in P. putida support the hypothesis that physicalseparation of the transcription- and translation-stimulating elementsleads to a far better improvement of protein production than achieved byidentifying mutations that simultaneously influence both processes. Italso means that the principle is not restricted to any particularbacterial host.

CONCLUSIONS

This study shows that mutations within 5′-UTRs that primarily stimulatetranscription or translation can be identified by library screening. Bythen physically separating these mutations within a re-designed 5′-UTRDNA region with extended length, we surprisingly demonstrated that astrongly improved expression output can be achieved. The applicabilityof already existing RBS design tools was also significantly improved bythis strategy, as poorly understood negative effects on transcriptioncan be avoided. The identification of 5′-UTR DNA elements specificallyenhancing transcription and the synergistic effect of this elementtogether with the effect of a translation-enhancing a 5′-UTR DNA onprotein production was unexpected. However, there can be a reasonableexpectation, based on the results disclosed herein, that the syntheticmRNA leaders can be applied to a wider range of genes than the twotested here. Accordingly, the present invention is universallyapplicable to improve recombinant expression, particularly in bacteria.

The invention claimed is:
 1. A method of enhancing expression of adesired gene product in a recombinant gene expression system, saidmethod comprising expressing said gene using a synthetic mRNA leaderwhich comprises from 5′ to 3′: (i) a first mRNA leader sequence element;(ii) a spacer region; and (iii) a second mRNA leader sequence element;wherein said first mRNA leader sequence element is a first mutated PmmRNA leader capable of enhancing transcription of a gene relative to anunmutated Pm mRNA leader sequence of SEQ ID NO:1, wherein the firstmutated Pm mRNA leader has at least 80% sequence identity to theunmutated Pm mRNA leader sequence of SEQ ID NO:1; and wherein saidsecond mRNA leader sequence element is a second mutated Pm mRNA leadercapable of enhancing the translation of a gene transcript relative to anunmutated Pm mRNA leader sequence of SEQ ID NO:1, wherein the secondmutated Pm mRNA leader has at least 80% sequence identity to theunmutated Pm mRNA leader sequence of SEQ ID NO:1.
 2. The method of claim1, wherein each mutated Pm mRNA leader sequence is generated byintroducing one or more mutations into the DNA corresponding to theunmutated Pm mRNA leader of SEQ ID NO:1, and selecting an mRNA leadermutant which enhances transcription of a gene and/or translation of agene transcript.
 3. The method of claim 1, wherein said spacer regioncomprises 4-200 nucleotides.
 4. The method of claim 1, wherein saidrecombinant gene expression system comprises a promoter selected fromany one of a Pm promoter, a Ptac promoter, a PtrcT7 RNA polymerasepromoter, λP_(L) or a P_(BAD) promoter.
 5. The method of claim 1,wherein each mutated Pm mRNA leader comprises 1 to 6 mutations.
 6. Themethod of claim 2, wherein in said second mutated Pm mRNA leader saidone or more mutations are present at or downstream of position +8 fromthe transcriptional start site.
 7. The method of claim 2, wherein insaid second mutated Pm mRNA leader said one or more mutations are notmade to the Shine-Dalgarno sequence.
 8. The method of claim 2, whereinin said second mutated Pm mRNA leader said one or more mutations do notinclude the insertion or creation of functional AU-rich sites.
 9. Themethod of claim 1, wherein a vector comprising a promoter, a DNA regioncorresponding to said synthetic mRNA leader and said gene encoding saiddesired gene product is introduced into a host cell and said host cellis cultured to allow said gene to be expressed.
 10. The method of claim2, wherein said mutations are selected from a substitution, a deletionor an addition, or a combination thereof.
 11. The method of claim 4,wherein said promoter is a Pm promoter.
 12. The method of claim 9,wherein said host cell is a prokaryotic cell.
 13. The method of claim 9,wherein said method further comprises a step of recovering, purifying,extracting or isolating the gene product expressed by said host cell.14. The method of claim 1, wherein the first mutated Pm mRNA leader isselected from any one of SEQ ID NOs: 18-23 and/or said second mutated PmmRNA leader is selected from any one of SEQ ID NOs: 25-46.
 15. Themethod of claim 1, wherein the first mutated Pm mRNA leader is selectedfrom any one of SEQ ID NOs: 21-23 and/or said second mutated Pm mRNAleader is selected from any one of SEQ ID NOs: 32, 41, 42 and
 45. 16.The method of claim 1, wherein the first mutated Pm mRNA leader is SEQID NO: 21 and said second mRNA leader is SEQ ID NO: 42.